Novel antibodies and uses thereof

ABSTRACT

The present invention provides an antibody or an antigen-binding fragment thereof with binding specificity for human interleukin-1 receptor accessory protein (IL1RAP) wherein the antibody or antigen-binding fragment is capable of inhibiting to domain 3 of human IL1RAP. The invention further provides the use of such antibodies or an antigen-binding fragments in the treatment and/or diagnosis of cancers, such as leukemias and melanoma.

This application is a 371 application of international application no.PCT/EP2015/068208, which was filed on Aug. 6, 2015, and claims priorityto United Kingdom application no. GB 1413913.3, which was filed on Aug.6, 2014, both of which are incorporated by reference in their entirety.

FIELD OF INVENTION

The present invention relates to antibody-based agents for the treatmentand diagnosis of diseases and conditions associated with an IL-1biomarker (specifically, IL1RAP) and/or responsive to inhibition of IL-1signalling. In particular, there are provided antibody-based agents forthe treatment and diagnosis of cancers, including but not limited toleukemias (such as chronic myeloid leukemia, acute myeloid leukemia,acute lymphoblastic leukemia, myeloproliferative disorders andmyelodysplastic syndrome) and cancers associated with solid tumourformation (such as melanoma, lung cancer, and breast cancer).

BACKGROUND

Interleukin-1 Biology

Interleukin-1 (IL-1) is a potent pro-inflammatory cytokine that can beproduced by a variety of cell types, including mononuclear phagocytes,in response to infection and inflammation. The IL-1 family consists ofseven agonists, including IL-1α and IL-1β, and three naturally occurringreceptor antagonists, including the IL-1 receptor antagonist (IL-1Ra)(Dinarello, C A, Blood 1996, 87(6): 2095-147). Two IL-1 receptors, IL-1Rtype I and IL-1R type II, have been identified. Both receptors caninteract with all three forms of the IL-1 family molecules. IL-1R1 isresponsible for mediating IL-1-induced cellular activation. However, theIL-1/IL-1RI complex cannot signal by itself, but is dependent onassociation with a second receptor chain, IL-1R Accessory Protein(IL1RAP) (Dinarello, C A, Blood 1996, 87(6): 2095-147). In contrast toIL-1RI, IL-1RII does not induce cellular activation upon binding to IL-1and thus IL-1RII functions as regulatory decoy receptor, leading to anet decrease in IL-1 available to bind to IL-1RI.

In addition to IL1-signaling, IL1RAP is critical for mediating theeffects of IL-33, through the ST2/IL1RAP complex, and IL36, through theIL1Rrp2/IL1RAP complex (Garlanda et al., Immunity. 2013 Dec. 12;39(6):1003-18)

IL-1 is a potent pro-inflammatory cytokine, which is induced at sites oflocal infection or inflammation and is involved in the regulation of avariety of physiological and cellular events (summarised in Dinarello CA, CHEST, 2000, 118: 503-508 and Dinarello, C A, Clin Exp Rheumatol,2002, 20(5 Suppl 27): S1-13). It is capable of activating several celltypes including leukocytes and endothelial cells. IL-1 induces andamplifies immunological responses by promoting the production andexpression of adhesion molecules, cytokines, chemokines and otherinflammatory mediators such as prostaglandin E₂ and nitric oxide (NO).As a consequence, local inflammation is amplified and sustained. Inaddition, the IL-1-induced production of inflammatory mediators resultsin fever, headache, hypotension and weight loss. Furthermore, IL-1 is ahematopoietic growth factor and has been shown to reduce the nadir ofleukocytes and platelets in patients during bone marrow transplantation.IL-1 has also been shown to promote angiogenesis by inducing theproduction of vascular endothelial growth factor, thereby promotingpannus formation and blood supply in rheumatic joints. Finally, IL-1 hasbeen shown to promote the bone and cartilage degradation in rheumaticdiseases.

The Role of IL-1 in Disease

IL-1 is implicated in a wide range of diseases and conditions rangingfrom gout to cancer (for reviews, see Dinarello et al., 2012, NatureReviews 11:633-652 and Dinarello, 2014, Mol. Med. 20(suppl. 1):S43-S58;the disclosures of which are incorporated herein by reference),including:

-   -   Joint, bone and muscle diseases, such as rheumatoid arthritis        and osteoarthritis;    -   Hereditary systemic autoinflammatory diseases, such as familial        Mediterranean fever;    -   Systemic autoinflammatory diseases, such as systemic juvenile        idiopathic arthritis and adult-onset Still's disease;    -   Common inflammatory diseases, such as gout and type 2 diabetes;    -   Acute-onset ischemic diseases, such as myocardial infarction;        and    -   Cancer.

A number of therapies for blocking IL-1 activity are approved and indevelopment. Targeting IL-1 began in 1993 with the introduction ofanakinra (Kineret; Amgen), a recombinant form of the naturally occurringIL-1 receptor antagonist (IL-Ra), which blocks the activity of bothIL-1α and IL-1β; this therapeutic has since been used to demonstrate arole for IL-1 in numerous diseases (see above). Anakinra currentlydominates the field of IL-1 therapeutics owing to its good safetyrecord, short half-life and multiple routes of administration.Neutralising IL-1 with antibodies or soluble receptors has also provedto be effective, and the soluble decoy receptor rilonacept (Arcalyst;Regeneron) and the anti-IL-1β neutralizing monoclonal antibodycanakinumab (Ilaris; Novartis) have now been approved for certain raregenetic conditions, such cryopyrin-associated periodic syndromes (CAPS).Other therapeutic approaches, including IL-1α neutralisation, atherapeutic vaccine targeting IL-1β and a chimaeric IL-1Ra, are in earlyclinical trials. In addition, orally active small-molecule inhibitors ofIL-1 production, such as caspase 1 inhibitors, have been developed andare being tested.

Interleukin-33 (IL-33) is a nuclear-associated cytokine of the IL-1family. IL-33 signals via the receptor IL-33R (ST2) and plays animportant role in allergy, asthma, infections, inflammation, and inpromoting cancer growth (see Cayrol & Girard (2014) Curr Opin Immunol.31:31-7 and Maywald et al. (2015) PNAS 112(19):E2487-2496). IL1RAP iscritical part of the IL-33R (ST2) receptor complex to convey the signalsby IL-33 (see Chackerian et al. (2007) J Immunol. 179(4):2551-2555).

IL1RAP as a Biomarker for Neoplastic Disorders

Tumour biomarkers are endogenous proteins or metabolites whose amountsor modifications are indicative of tumour state, progressioncharacteristics, and response to therapies. They are present in tumourtissues or body fluids and encompass a wide variety of molecules,including transcription factors, cell surface receptors, and secretedproteins. Effective tumour markers are in great demand since they havethe potential to reduce cancer mortality rates by facilitating diagnosisof cancers at early stages and by helping to individualize treatments.During the last decade, improved understanding of carcinogenesis andtumour progression has revealed a large number of potential tumourmarkers. It is predicted that even more will be discovered in the nearfuture with the application of current technologies such as tissuemicroarrays, antibody arrays, and mass spectrometry.

Interleukin-1 receptor accessory protein (IL1RAP) has previously beenidentified as cell-surface biomarker associated with haematologicalneoplastic disorders such as chronic myeloid leukemia (CML), acutemyeloid leukemia (AML) and myelodysplatic syndromes (MDS) (for example,see WO 2011/021014 to Cantargia A B, Järås et al., 2010, Proc Natl AcadSci USA 107(37):16280-5, Askmyr et al., 2013, Blood. 121(18):3709-13 andBarreyro et al., 2012, Blood 120(6):1290-8, the disclosures of which areincorporated herein by reference). More recently, the usefulness ofIL1RAP as a diagnostic and therapeutic biomarker for solid tumours, suchas melanomas, has also been revealed (see WO 2012/098407 to CantargiaAB, the disclosures of which are incorporated herein by reference).

SUMMARY OF INVENTION

The present inventors have developed new anti-IL1RAP antibodies withimproved properties making them suitable for diagnosis and treatment ofdiseases and conditions associated with the IL1RAP biomarker and/orresponsive to inhibition of IL-1 signalling, IL-33 signalling and/orIL-36 signalling.

Accordingly, in a first aspect, the present invention provides anantibody or an antigen-binding fragment thereof (‘antibodypolypeptides’) with binding specificity for domain 3 of interleukin-1receptor accessory protein (‘IL1RAP’).

By “interleukin-1 receptor accessory protein”, “IL1RAP” and “IL1-RAP” wespecifically include the human IL1RAP protein, for example as describedin GenBank Accession No. AAB84059, NCBI Reference Sequence: NP_002173.1and UniProtKB/Swiss-Prot Accession No. Q9NPH3-1 (see also Huang et al.,1997, Proc. Natl. Acad. Sci. USA. 94 (24), 12829-12832, the disclosuresof which are incorporated herein by reference). IL1RAP is also known inthe scientific literature as IL1R3, C3orf13, FLJ37788, IL-1RAcP andEG3556.

By “domain 3” of IL1RAP we include the structural region defined byamino acids 235 to 369 of IL1RAP according to numbering used inAccession No. Q9NPH3 of UniProtKB/Swiss-Prot (see also Wang et al.,2010, Nature Immunology, 11:905-912, the disclosures of which areincorporated herein by reference). For example, the epitope to which theantibody or antigen-binding fragment binds may be located within aminoacids 235 to 239, 240 to 249, 250 to 259, 260 to 269, 270 to 279, 280 to289, 290 to 299, 300 to 309, 310 to 319, 320 to 329, 330 to 229, 240 to349, 350 to 359 or between amino acids 360 to 369 of IL1RAP.

Thus, the antibody polypeptides of the invention have specificity forIL1RAP. By “specificity” we mean that the antibody polypeptide iscapable of binding to IL1RAP in vivo, i.e. under the physiologicalconditions in which IL1RAP exists within the human body. Preferably, theantibody polypeptide does not bind to any other protein in vivo. Suchbinding specificity may be determined by methods well known in the art,such as ELISA, immunohistochemistry, immunoprecipitation, Western blotsand flow cytometry using transfected cells expressing IL1RAP.Advantageously, the antibody polypeptide is capable of bindingselectively to IL1RAP, i.e. it bind at least 10-fold more strongly toIL1RAP than to any other proteins.

By “an antibody or an antigen-binding fragment thereof” we includesubstantially intact antibody molecules, as well as chimaericantibodies, humanised antibodies, isolated human antibodies, singlechain antibodies, bispecific antibodies, antibody heavy chains, antibodylight chains, homodimers and heterodimers of antibody heavy and/or lightchains, and antigen-binding fragments and derivatives of the same.Suitable antigen-binding fragments and derivatives include Fv fragments(e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments(e.g. Fab fragments, Fab′ fragments and F(ab)₂ fragments), singlevariable domains (e.g. V_(H) and V_(L) domains) and domain antibodies(dAbs, including single and dual formats [i.e. dAb-linker-dAb]). Thepotential advantages of using antibody fragments, rather than wholeantibodies, are several-fold. The smaller size of the fragments may leadto improved pharmacological properties, such as better penetration ofsolid tissue. Moreover, antigen-binding fragments such as Fab, Fv, ScFvand dAb antibody fragments can be expressed in and secreted from E.coli, thus allowing the facile production of large amounts of the saidfragments.

Thus, in one embodiment, the antibody polypeptide of the inventioncomprises or consists of an intact antibody (such as an IgG1 antibody).

In an alternative embodiment, the antibody polypeptide of the inventioncomprises or consists of an antigen-binding fragment selected from thegroup consisting of Fv fragments (e.g. single chain Fv anddisulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab′fragments and F(ab)₂ fragments) and domain antibodies (e.g. single V_(H)variable domains or V_(L) variable domains).

The phrase “an antibody or an antigen-binding fragment thereof” is alsointended to encompass antibody mimics (for example, non-antibodyscaffold structures that have a high degree of stability yet allowvariability to be introduced at certain positions). Those skilled in theart of biochemistry will be familiar with many such molecules, asdiscussed in Gebauer & Skerra, 2009, Curr Opin Chem Biol 13(3): 245-255(the disclosures of which are incorporated herein by reference).Exemplary antibody mimics include: affibodies (also called Trinectins;Nygren, 2008, FEBS J, 275, 2668-2676); CTLDs (also called Tetranectins;Innovations Pharmac. Technol. (2006), 27-30); adnectins (also calledmonobodies; Meth. Mol. Biol., 352 (2007), 95-109); anticalins (DrugDiscovery Today (2005), 10, 23-33); DARPins (ankyrins; Nat. Biotechnol.(2004), 22, 575-582); avimers (Nat. Biotechnol. (2005), 23, 1556-1561);microbodies (FEBS J, (2007), 274, 86-95); peptide aptamers (Expert.Opin. Biol. Ther. (2005), 5, 783-797); Kunitz domains (J. Pharmacol.Exp. Ther. (2006) 318, 803-809); affilins (Trends. Biotechnol. (2005),23, 514-522); affimers (Avacta Life Sciences, Wetherby, UK).

Also included within the scope of the invention are chimaeric T-cellreceptors (also known as chimaeric T cell receptors, chimaericimmunoreceptors, and chimaeric antigen receptors or CARs) (see Pule etal., 2003, Cytotherapy 5(3):211-26, the disclosures of which areincorporated herein by reference). These are engineered receptors, whichgraft an arbitrary specificity onto an immune effector cell. Typically,CARs are used to graft the specificity of a monoclonal antibody onto a Tcell; with transfer of their coding sequence facilitated by retroviralvectors. The most common form of such molecules is fusions comprising asingle-chain variable fragment (scFv) derived from a monoclonal antibodyfused to CD3-zeta transmembrane and endodomain. When T cells expressthis fusion molecule, they recognize and kill target cells that expressthe transferred monoclonal antibody specificity.

Persons skilled in the art will further appreciate that the inventionalso encompasses modified versions of antibodies and antigen-bindingfragments thereof, whether existing now or in the future, e.g. modifiedby the covalent attachment of polyethylene glycol or another suitablepolymer (see below).

Methods of generating antibodies and antibody fragments are well knownin the art. For example, antibodies may be generated via any one ofseveral methods which employ induction of in vivo production of antibodymolecules, screening of immunoglobulin libraries (Orlandi. et al, 1989.Proc. Natl. Acad. Sci. U.S.A. 86:3833-3837; Winter et al., 1991, Nature349:293-299, the disclosures of which are incorporated herein byreference) or generation of monoclonal antibody molecules by cell linesin culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the Epstein-Barrvirus (EBV)-hybridoma technique (Kohler et al., 1975. Nature256:4950497; Kozbor et al., 1985. J. Immunol. Methods 81:31-42; Cote etal., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole et al., 1984.Mol. Cell. Biol. 62:109-120, the disclosures of which are incorporatedherein by reference).

Suitable methods for the production of monoclonal antibodies are alsodisclosed in “Monoclonal Antibodies: A manual of techniques”, H Zola(CRC Press, 1988, the disclosures of which are incorporated herein byreference) and in “Monoclonal Hybridoma Antibodies: Techniques andApplications”, J G R Hurrell (CRC Press, 1982, the disclosures of whichare incorporated herein by reference).

Likewise, antibody fragments can be obtained using methods well known inthe art (see, for example, Harlow & Lane, 1988, “Antibodies: ALaboratory Manual”, Cold Spring Harbor Laboratory, New York, thedisclosures of which are incorporated herein by reference). For example,antibody fragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment. Alternatively,antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods.

In one embodiment, the antibody polypeptides of the invention aredefined by reference to the variable regions of a murine-derivedantibody, designated ‘CAN01’, which comprises:

-   -   (a) a heavy chain variable region having the amino acid sequence        of SEQ ID NO: 1:

[SEQ ID NO: 1] Q V Q L Q Q S G T E L M K P G A S V K I S C K AT G Y T V S S Y W I D W V K Q T P G H G L E W IG E I L P G S A I N N Y N E K F K G K A T F T A DT S S N T A Y M Q L S S L T S E D S A V Y Y C AS G D Y F D S T F V Y Y W G Q G T T L T V S Sand

-   -   (b) a light chain variable region having the amino acid sequence        of SEQ ID NO: 2:

[SEQ ID NO: 2] D V L M T Q T P L S L P V S L G D Q A S I S C R SS Q S I V H S N G N T Y L E W Y L Q K P G Q S P K L L I Y K V S N R F S G V P D R F S G S G S G TD F T L K I S R V E A E D L G V Y Y C F Q G S H VP R T F G G G T K L E I K R

The term “amino acid” as used herein includes the standard twentygenetically-encoded amino acids and their corresponding stereoisomers inthe ‘D’ form (as compared to the natural ‘L’ form), omega-amino acidsother naturally-occurring amino acids, unconventional amino acids (e.g.α,α-disubstituted amino acids, N-alkyl amino acids, etc.) and chemicallyderivatised amino acids (see below).

When an amino acid is being specifically enumerated, such as “alanine”or “Ala” or “A”, the term refers to both L-alanine and D-alanine unlessexplicitly stated otherwise. Other unconventional amino acids may alsobe suitable components for polypeptides of the present invention, aslong as the desired functional property is retained by the polypeptide.For the peptides shown, each encoded amino acid residue, whereappropriate, is represented by a single letter designation,corresponding to the trivial name of the conventional amino acid.

In one embodiment, the antibody polypeptides as defined herein compriseor consist of L-amino acids.

It will be appreciated by persons skilled in the art that any intact IgGantibody comprising the above variable regions may be used as thereference antibody to identify antibody polypeptides of the inventionthat competitively inhibit CAN01 binding to IL1RAP.

Thus, in one embodiment, the CAN01 antibody used as a reference todetermine competitive binding is an intact IgG antibody comprising:

-   -   (a) a heavy chain comprising a variable domain of SEQ ID NO:1        grafted on to an murine IgG1 or IgG2a constant region; and    -   (b) a light chain comprising a variable domain of SEQ ID NO:2        grafted on to a murine kappa constant region.

Alternatively, the reference antibody may be a chimaeric, intact IgGantibody comprising:

-   -   (a) a heavy chain comprising a variable domain of SEQ ID NO:1        grafted on to an human IgG1 constant region (for example, such        as SEQ ID NO: 30; as encoded by the pFUSEss-CHIg-hG1 vector        InvivoGen, San Diego, USA); and    -   (b) a light chain comprising a variable domain of SEQ ID NO:2        grafted on to a human kappa constant region (for example, such        as SEQ ID NO: 29; as encoded by the pFUSE2ss-CLIg-hk vector        InvivoGen, San Diego, USA).

Suitable methods for determining whether a given test antibody is ableto inhibit the binding of a reference antibody to an antigen are wellknown in the art. For example, to test whether a test antibody is ableto inhibit the binding of the CAN01 reference antibody to a cell surfaceantigen, cells expressing the antigen can be pre-incubated with the testantibody for 20 min before cells are washed and incubated with thereference CAN01 antibody conjugated to a fluorophore, which can bedetected by flow cytometry. If the pre-incubation with the test antibodyreduces the detection of the reference CAN01 antibody in flow cytometry,the test antibody inhibits the binding of the reference antibody to thecell surface antigen.

Such competitive binding inhibition may also be determined using BIAcorechips with immobilised IL1RAP and incubating with the reference antibody(such as ‘CAN01’) with and without an antibody polypeptide to be tested.Alternatively, a pair-wise mapping approach can be used, in which thereference antibody is immobilised to the surface of the BIAcore chip,IL1RAP antigen is bound to the immobilised antibody, and then a secondantibody is tested for simultaneous IL1 RAP-binding ability (see‘BIAcore Assay Handbook’, GE Healthcare Life Sciences, 29-0194-00 AA May2012; the disclosures of which are incorporated herein by reference).

In a further alternative, competitive binding inhibition can bedetermined using flow cytometry. For example, to test whether a testantibody is able to inhibit the binding of the reference antibody to acell surface antigen, cells expressing the antigen can be pre-incubatedwith the test antibody for 20 min before cells are washed and incubatedwith the reference antibody conjugated to a fluorophore, which can bedetected by flow cytometry. If the pre-incubation with the test antibodyreduces the detection of the reference antibody in flow cytometry, thetest antibody inhibits the binding of the reference antibody to the cellsurface antigen. If the antibody to be tested exhibits high affinity forIL1RAP, then a reduced pre-incubation period may be used (or even nopre-incubation at all).

In a further alternative, competitive binding inhibition can bedetermined using an ELISA (e.g. as described in Example N).

Competitive binding typically arises because the test antibody binds at,or at least very close to, the epitope on the antigen to which binds thereference antibody (in this case, CAN01). However, it will beappreciated by persons skilled in the art that competitive binding mayalso arise by virtue of steric interference; thus, the test antibody maybind at an epitope different from that to which the reference antibodybinds but may still be of sufficient size or configuration to hinder thebinding of the reference antibody to the antigen.

The antibodies and antigen-binding fragments of the present inventionwere identified after extensive screening of a large number ofanti-IL1RAP antibodies, on the basis of exhibiting properties that makethem particularly suitable as diagnostic and therapeutic agents forcancer.

Thus, in one embodiment, the antibody or antigen-binding fragmentexhibits one or more of the following properties:

-   -   (a) a binding affinity (K_(D)) for human IL1RAP of 2 nM or        greater, i.e. the K_(D)≦2 nM (for example, as determined using        the Biacore methodology in Example A);    -   (b) cross-reactivity with IL1RAP from Macaca fascicularis (for        example, as determined in Example D);    -   (c) an inhibitory action on IL-1 signalling (for example, as        determined in Example E, L and M);    -   (d) capability of inducing antibody-dependent cell-mediated        cytotoxicity (ADCC) in one or more cancer cell lines (such as a        CML, AML, ALL and/or melanoma cell lines, and/or cell line        models of one or more of the other cancer types identified        below) (for example, as determined in Examples F and G); and/or    -   (e) capability of internalisation upon binding to one or more        cancer cell lines (such as a CML, AML, ALL and/or melanoma cell        lines, and/or cell line models of one or more of the other        cancer types identified below) (for example, as determined in        Example H).

Advantageously, the antibody or antigen-binding fragment exhibits all ofthe above properties.

In one embodiment, the antibody or antigen-binding fragment furtherexhibits an inhibitory action on IL-33 signalling and/or IL-36signalling (for example, as determined in Examples L and M below).

Optionally, the antibody or antigen-binding fragment does not exhibit aninhibitory action on one or more of IL-1 signalling, IL-33 signallingand IL-36 signalling (for example, as determined in Example E, L and M).For example, the antibody may be CAN01, which lacks any appreciableinhibitory action on IL-1 signalling or IL-33 signalling (see Examples Land M below).

In an alternative embodiment, the antibody or antigen-binding fragmentexhibits one or more of properties (a), (b), (c) and (e) above, but isnot capable of inducing ADCC.

In one embodiment, the antibody or antigen-binding fragment is capableof binding to an epitope on the extracellular domain of IL1RAP whichoverlaps, at least in part, with the epitope on IL1RAP to whichreference antibody CAN01 is capable of binding. Thus, the antibody orantigen-binding fragment may be capable of binding to an epitope locatedat/within domain 3 of IL1RAP, i.e. amino acids 235 to 367 (see above).

In a preferred embodiment, the antibody or antigen-binding fragmentthereof according to the first aspect of the invention comprises a heavychain variable region comprising the following CDRs:

-   -   a) G Y T V S S Y [SEQ ID NO: 3] or an amino acid sequence having        at least 60% sequence identity therewith, for example at least        70%, 80%, or 90% sequence identity;    -   b) L P G S A I [SEQ ID NO: 4] or an amino acid sequence having        at least 60% sequence identity therewith, for example at least        70%, 80%, or 90% sequence identity; and    -   c) G D Y F D S T F V Y Y [SEQ ID NO: 5] or an amino acid        sequence having at least 60% sequence identity therewith, for        example at least 70%, 80%, or 90% sequence identity.

Thus, the antibody or antigen-binding fragment thereof may comprise aheavy chain variable region comprising the CDRs of SEQ ID NOs 3, 4 and5.

For example, the antibody or antigen-binding fragment thereof maycomprise a heavy chain variable region having the amino acid sequence ofthe corresponding region of the CAN01 reference antibody, i.e. SEQ IDNO:1.

However, it will be appreciated that a low level of mutation (typically,just one or two amino acids) within a CDR sequence may be toleratedwithout loss of the specificity of the antibody or antigen-bindingfragment for IL1RAP.

Percent identity can be determined by, for example, the LALIGN program(Huang and Miller, Adv. Appl. Math. (1991) 12:337-357, the disclosuresof which are incorporated herein by reference) at the Expasy facilitysite (http://www.ch.embnet.org/software/LALIGN_form.html) using asparameters the global alignment option, scoring matrix BLOSUM62, openinggap penalty—14, extending gap penalty—4. Alternatively, the percentsequence identity between two polypeptides may be determined usingsuitable computer programs, for example the GAP program of theUniversity of Wisconsin Genetic Computing Group and it will beappreciated that percent identity is calculated in relation topolypeptides whose sequence has been aligned optimally.

The alignment may alternatively be carried out using the Clustal Wprogram (as described in Thompson et al., 1994, Nucl. Acid Res.22:4673-4680, which is incorporated herein by reference). The parametersused may be as follows:

-   -   Fast pair-wise alignment parameters: K-tuple(word) size; 1,        window size; 5, gap penalty; 3, number of top diagonals; 5.        Scoring method: x percent.    -   Multiple alignment parameters: gap open penalty; 10, gap        extension penalty; 0.05.    -   Scoring matrix: BLOSUM.

Alternatively, the BESTFIT program may be used to determine localsequence alignments.

In a further preferred embodiment, the antibody or antigen-bindingfragment thereof according to the first aspect of the inventioncomprises a heavy chain variable region comprising the following CDRs:

-   -   a) G Y T V S S Y W I D [SEQ ID NO: 6] or an amino acid sequence        having at least 60% sequence identity therewith, for example at        least 70%, 80%, or 90% sequence identity;    -   b) E I L P G S A I N N Y N E K F K G [SEQ ID NO:7] or an amino        acid sequence having at least 60% sequence identity therewith,        for example at least 70%, 80%, or 90% sequence identity; and    -   c) G D Y F D S T F V Y Y [SEQ ID NO: 5] or an amino acid        sequence having at least 60% sequence identity therewith, for        example at least 70%, 80%, or 90% sequence identity.

For example, the antibody polypeptide may comprise a heavy chainvariable region comprising the CDRs of SEQ ID NOs 6, 7 and 5.

As indicated above, the CAN01 reference antibody is a murine antibody.However, the component heavy and light chains of this antibody may behumanised in order to produce antibody polypeptides more suitable foruse in humans, e.g. due to their reduced immunogenicity. For example,the CDRs of SEQ ID NOs 3, 4 and 5 (or the CDRs of SEQ ID NOs 6, 7 and 5)may be engrafted into a human variable region framework.

It will be appreciated by persons skilled in the art that for humantherapy or diagnostics, human or humanised antibodies are preferablyused. Humanised forms of non-human (e.g. murine) antibodies aregenetically engineered chimaeric antibodies or antibody fragments havingpreferably minimal-portions derived from non-human antibodies. Humanisedantibodies include antibodies in which complementary determining regionsof a human antibody (recipient antibody) are replaced by residues from acomplementary determining region of a non-human species (donor antibody)such as mouse, rat of rabbit having the desired functionality. In someinstances, Fv framework residues of the human antibody are replaced bycorresponding non-human residues. Humanised antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported complementarity determining region or framework sequences. Ingeneral, the humanised antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the complementarity determining regions correspondto those of a non-human antibody and all, or substantially all, of theframework regions correspond to those of a relevant human consensussequence. Humanised antibodies optimally also include at least a portionof an antibody constant region, such as an Fc region, typically derivedfrom a human antibody (see, for example, Jones et al., 1986. Nature321:522-525; Riechmann et al., 1988, Nature 332:323-329; Presta, 1992,Curr. Op. Struct. Biol. 2:593-596, the disclosures of which areincorporated herein by reference).

Methods for humanising non-human antibodies are well known in the art.Generally, the humanised antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues, often referred to as imported residues, aretypically taken from an imported variable domain. Humanisation can beessentially performed as described (see, for example, Jones et al.,1986, Nature 321:522-525; Reichmann et al., 1988. Nature 332:323-327;Verhoeyen et al., 1988, Science 239:1534-15361; U.S. Pat. No. 4,816,567,the disclosures of which are incorporated herein by reference) bysubstituting human complementarity determining regions withcorresponding rodent complementarity determining regions. Accordingly,such humanised antibodies are chimaeric antibodies, whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanised antibodies may be typically human antibodies inwhich some complementarity determining region residues and possibly someframework residues are substituted by residues from analogous sites inrodent antibodies.

Human antibodies can also be identified using various techniques knownin the art, including phage display libraries (see, for example,Hoogenboom & Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J.Mol. Biol. 222:581; Cole et al., 1985, In: Monoclonal antibodies andCancer Therapy, Alan R. Liss, pp. 77; Boerner et al., 1991. J. Immunol.147:86-95, the disclosures of which are incorporated herein byreference).

Thus, the antibody or antigen-binding fragment thereof of the inventionmay be humanised, for example it may comprise a heavy chain variableregion having one of the following amino acid sequence of any one of SEQID NOs: 8 to 10 or an amino acid sequence having at least 90% sequenceidentity therewith:

-   -   a)

[SEQ ID NO: 8] E V Q L V Q S G A E V K K P G A T V K I S C KA S G Y T V S S Y W I D W V R Q A P G Q G L EW M G E I L P G S A I N N Y A E K F Q G R V TF T A D T S T D T A Y M E L S S L R S E D T A VY Y C A S G D Y F D S T F V Y Y W G Q G T T V T V S S;

-   -   b)

[SEQ ID NO: 9] Q V Q L V Q S G A E V K K P G A T V K I S C KA S G Y T V S S Y W I D W V R Q A P G Q G L EW M G E I L P G S A I T N Y A E K F Q G R V TF T A D T S T S T A Y M E L S S L R S E D T A VY Y C A S G D Y F D S T F V Y Y W G Q G T T V  T V S S;or

-   -   c)

[SEQ ID NO: 10] Q V Q L V Q S G A E V K K P G A T V K I S C KA D G Y T V S S Y W I D W V R Q A P G Q G L EW M G E I L P G S A I T N Y A E K F Q G R V TF T A D T S T S T A Y M E L S S L R S E D T A TY Y C A S G D Y F D S T F V Y Y W G Q G T T V T V S S.

For example, the antibody or antigen-binding fragment thereof maycomprise a heavy chain variable region having an amino acid sequence ofany one of SEQ ID NOs: 8 to 10.

In a related preferred embodiment, the antibody or antigen-bindingfragment thereof comprises a light chain variable region comprising thefollowing CDRs:

-   -   a) R S S Q S I V H S N G N T Y L E [SEQ ID NO: 11] or an amino        acid sequence having at least 60% sequence identity therewith,        for example at least 70%, 80%, or 90% sequence identity;    -   b) K V S N R F S [SEQ ID NO: 12] or an amino acid sequence        having at least 60% sequence identity therewith, for example at        least 70%, 80%, or 90% sequence identity; and    -   c) F Q G S H V P R T [SEQ ID NO: 13] or an amino acid sequence        having at least 60% sequence identity therewith, for example at        least 70%, 80%, or 90% sequence identity.

Thus, the antibody or antigen-binding fragment thereof may comprise alight chain variable region comprising the CDRs of SEQ ID NOs 11, 12 and13.

For example, the antibody or antigen-binding fragment thereof maycomprise a light chain variable region having the amino acid sequence ofthe corresponding region of the murine CAN01 reference antibody, i.e.SEQ ID NO:2.

As in the case of the heavy chain variable region detailed above, itwill be appreciated that the light chain variable region of the antibodypolypeptide of the invention may be humanised in order to produce agentsmore suitable for use in humans. For example, the CDRs of SEQ ID NOs 11,12 and 13 may be engrafted into a human variable region framework.

Thus, the antibody or antigen-binding fragment thereof may comprise alight chain variable region which comprises or consists of the aminoacid sequence of any one of SEQ ID NOs: 14 to 18 or an amino acidsequence having at least 90% sequence identity therewith:

-   -   a)

[SEQ ID NO: 14] D I V M T Q S P L S L P V T P G E P A S I S C RS S Q S I V H S N G N T Y L E W Y L Q K P G QS P Q L L I Y K V S N R F S G V P D R F S G SG S G T D F T L K I S R V E A E D V G V Y Y C FQ G S H V P R T F G G G T K V E I K R;

-   -   b)

[SEQ ID NO: 15] D I V M T Q S P L S L P V T P G Q P A S I S C RS S Q S I T H S N G N T Y L E W Y L Q K P G Q SP Q L L I Y K V S N R D S G V P D R F S G S G SG T D F T L K I S R V E A E D V G V Y Y C F QG S H V P R T F G G G T K V E I K R;

-   -   c)

[SEQ ID NO: 16] D I V M T Q S P L S L P V T P G E P A S I S C RS S Q S I T H S N G Q T Y L E W Y L Q K P G QS P Q L L I Y K V S N R A S G V P D R F S G SG S G T D F T L K I S R V E A E D V G V Y Y C FQ G S H V P R T F G G G T K V E I K R;

-   -   d)

[SEQ ID NO: 17] D I V M T Q S P L S L P V T P G E P A S I S C RS S Q S I T H S S G N T Y L E W Y L Q K P G Q SP Q L L I Y K V S N R A S G V P D R F S G S GS G T D F T L K I S R V E A E D V G V Y Y C F QG S H V P R T F G G G T K V E I K R;or

-   -   e)

[SEQ ID NO: 18] P Q L L I Y K V S N R D S G V P D R F S G S GS G T D F T L K I S R V E A E D V G V Y Y C F QG S H V P R T F G G G T K V E I K R.

For example, the antibody or antigen-binding fragment thereof maycomprise a light chain variable region having an amino acid sequence ofany one of SEQ ID NOs: 14 to 18.

In one embodiment, the antibody or antigen-binding fragment thereofcomprises a murine heavy chain variable region which comprises orconsists of the amino acid sequence of any one of SEQ ID NO: 1 and amurine light chain variable region which comprises or consists of theamino acid sequence of any one of SEQ ID NO: 2.

Alternatively, the antibody or antigen-binding fragment thereof maycomprise a humanised heavy chain variable region which comprises orconsists of the amino acid sequence of any one of SEQ ID NOs: 8 to 10and a humanised light chain variable region which comprises or consistsof the amino acid sequence of any one of SEQ ID NOs: 14 to 18.

For example, the antibody or antigen-binding fragment thereof maycomprise:

-   -   a) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 8 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 14;    -   b) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 9 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 14;    -   c) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 10 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 14;    -   b) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 8 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 15;    -   d) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 9 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 15;    -   e) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 10 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 15;    -   c) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 8 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 16;    -   f) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 9 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 16;    -   g) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 10 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 16;    -   d) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 8 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 17;    -   h) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 9 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 17;    -   i) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 10 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 17;    -   e) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 8 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 18;    -   j) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 9 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 18; or    -   k) a heavy chain variable region which comprises or consists of        the amino acid sequence of SEQ ID NO: 10 and a light chain        variable region which comprises or consists of the amino acid        sequence of SEQ ID NO: 18.

In an alternative embodiment, the antibody polypeptides of the inventionare defined by reference to the variable regions of a murine-derivedantibody, designated ‘CAN03’, which comprises:

-   -   (a) a heavy chain variable region having the amino acid sequence        of SEQ ID NO: 19:

[SEQ ID NO: 19] D V K L V E S G G G L V K P G G S L K L S C A AS G F T F S I Y T M S W V R Q T P E K R L E W VA T I S I G G S Y I N Y P D S V K G R F T I S R DN A K N T L Y L Q M S S L K S E D T A I Y Y C S RE V D G S Y A M D Y W G Q G T S V T V S Sand

-   -   (b) a light chain variable region having the amino acid sequence        of SEQ ID NO: 20:

[SEQ ID NO: 20] D I L L T Q S P A I L S V S P G E R V S F S C R AS Q S I G T S I H W Y Q R R T N G S P R L L I K SA S E S I S G I P S R F S G S G S G T D F T L S IN S V E S E D I A D Y Y C Q Q S N S W P T T F G A G T K L E L K R

It will be appreciated by persons skilled in the art that any intact IgGantibody comprising the above variable regions may be used as thereference antibody to identify antibody polypeptides of the inventionthat competitively inhibit CAN03 binding to IL1RAP (as described abovein relation to the use of CAN01 as a reference antibody).

Thus, in one embodiment, the CAN03 antibody used as a reference todetermine competitive binding is an intact IgG antibody comprising:

-   -   (a) a heavy chain comprising a variable domain of SEQ ID NO:19        grafted on to an murine IgG1 or IgG2a constant region; and    -   (b) a light chain comprising a variable domain of SEQ ID NO:20        grafted on to a murine kappa constant region.

Alternatively, the reference antibody may be a chimaeric, intact IgGantibody comprising:

-   -   (a) a heavy chain comprising a variable domain of SEQ ID NO:19        grafted on to an human IgG1 constant region (for example, such        as SEQ ID NO:30; as encoded by the pFUSEss-CHIg-hG1 vector        InvivoGen, San Diego, USA); and    -   (b) a light chain comprising a variable domain of SEQ ID NO:20        grafted on to a human kappa constant region (for example, such        as SEQ ID NO:29; as encoded by the pFUSE2ss-CLIg-hk vector        InvivoGen, San Diego, USA).

Suitable methods for determining whether a given test antibody is ableto inhibit the binding of a reference antibody to an antigen are wellknown in the art (see above).

In one embodiment, the antibody or antigen-binding fragment whichcompetitively inhibits CAN03-binding to IL1RAP exhibits one or more ofthe following properties:

-   -   (a) a binding affinity (K_(D)) for human IL1RAP of 500 pM or        greater, i.e. the K_(D)≦500 pM (for example, as determined using        the Biacore methodology in Example A);    -   (b) cross-reactivity with IL1RAP from Macaca fascicularis (for        example, as determined in Example D);    -   (c) an inhibitory action on IL-1 signalling (for example, as        determined in Example E);    -   (d) capability of inducing antibody-dependent cell-mediated        cytotoxicity (ADCC) in one or more cancer cell lines (such as a        CML, AML, ALL and/or melanoma cell lines, and/or cell line        models of one or more of the other cancer types identified        below) (for example, as determined in Examples F and G); and/or    -   (e) capability of internalisation upon binding to one or more        cancer cell lines (such as a CML, AML, ALL and/or melanoma cell        lines, and/or cell line models of one or more of the other        cancer types identified below) (for example, as determined in        Example H).

Advantageously, the antibody or antigen-binding fragment exhibits all ofthe above properties.

In one embodiment, the antibody or antigen-binding fragment furtherexhibits an inhibitory action on IL-33 signalling and/or IL-36signalling (for example, as determined in Examples L and M below).

In one embodiment, the antibody or antigen-binding fragment is capableof binding to an epitope on the extracellular domain of IL1RAP whichoverlaps, at least in part, with the epitope on IL1RAP to whichreference antibody CAN03 is capable of binding. Thus, the antibody orantigen-binding fragment may be capable of binding to an epitope locatedat/within domain 3 of IL1RAP, i.e. amino acids 235 to 367 (see above).

In a preferred embodiment, the antibody or antigen-binding fragmentthereof according to the first aspect of the invention comprises a heavychain variable region comprising the following CDRs:

-   -   a) G F T F S I Y [SEQ ID NO: 21] or an amino acid sequence        having at least 60% sequence identity therewith, for example at        least 70%, 80%, or 90% sequence identity;    -   b) S I G G S Y [SEQ ID NO: 22] or an amino acid sequence having        at least 60% sequence identity therewith, for example at least        70%, 80%, or 90% sequence identity; and    -   c) E V D G S Y A M D Y [SEQ ID NO: 23] or an amino acid sequence        having at least 60% sequence identity therewith, for example at        least 70%, 80%, or 90% sequence identity.

Thus, the antibody or antigen-binding fragment thereof may comprise aheavy chain variable region comprising the CDRs of SEQ ID NOs 21, 22 and23.

For example, the antibody or antigen-binding fragment thereof maycomprise a heavy chain variable region having the amino acid sequence ofthe corresponding region of the CAN01 reference antibody, i.e. SEQ IDNO:19.

However, it will be appreciated that a low level of mutation (typically,just one or two amino acids) within a CDR sequence may be toleratedwithout loss of the specificity of the antibody or antigen-bindingfragment for IL1RAP.

In a further preferred embodiment, the antibody or antigen-bindingfragment thereof according to the first aspect of the inventioncomprises a heavy chain variable region comprising the following CDRs:

-   -   a) G F T F S I Y T M S [SEQ ID NO: 24] or an amino acid sequence        having at least 60% sequence identity therewith, for example at        least 70%, 80%, or 90% sequence identity;    -   b) T I S I G G S Y I D N Y P D S V K G [SEQ ID NO: 25] or an        amino acid sequence having at least 60% sequence identity        therewith, for example at least 70%, 80%, or 90% sequence        identity; and    -   c) E V D G S Y A M D Y [SEQ ID NO: 23] or an amino acid sequence        having at least 60% sequence identity therewith, for example at        least 70%, 80%, or 90% sequence identity.

For example, the antibody polypeptide may comprise a heavy chainvariable region comprising the CDRs of SEQ ID NOs 24, 25 and 23.

As indicated above, the CAN03 reference antibody is a murine antibody.However, the component heavy and light chains of this antibody may behumanised in order to produce antibody polypeptides more suitable foruse in humans, e.g. due to their reduced immunogenicity. For example,the CDRs of SEQ ID NOs 21, 22 and 23 (or the CDRs of SEQ ID NOs 24, 25and 23) may be engrafted into a human variable region framework.

In a related preferred embodiment, the antibody or antigen-bindingfragment thereof comprises a light chain variable region comprising thefollowing CDRs:

-   -   a) R A S Q S I G T S I H [SEQ ID NO: 26] or an amino acid        sequence having at least 60% sequence identity therewith, for        example at least 70%, 80%, or 90% sequence identity;    -   b) S A S E S I S [SEQ ID NO: 27] or an amino acid sequence        having at least 60% sequence identity therewith, for example at        least 70%, 80%, or 90% sequence identity; and    -   c) Q Q S N S W P T T [SEQ ID NO: 28] or an amino acid sequence        having at least 60% sequence identity therewith, for example at        least 70%, 80%, or 90% sequence identity

Thus, the antibody or antigen-binding fragment thereof may comprise alight chain variable region comprising the CDRs of SEQ ID NOs 26, 27 and28.

For example, the antibody or antigen-binding fragment thereof maycomprise a light chain variable region having the amino acid sequence ofthe corresponding region of the murine CAN01 reference antibody, i.e.SEQ ID NO:20.

As in the case of the heavy chain variable region detailed above, itwill be appreciated that the light chain variable region of the antibodypolypeptide of the invention may be humanised in order to produce agentsmore suitable for use in humans. For example, the CDRs of SEQ ID NOs 26,27 and 28 may be engrafted into a human variable region framework.

In one embodiment, the antibody or antigen-binding fragment thereofcomprises a murine heavy chain variable region which comprises orconsists of the amino acid sequence of any one of SEQ ID NO: 19 and amurine light chain variable region which comprises or consists of theamino acid sequence of any one of SEQ ID NO: 20.

Alternatively, the antibody or antigen-binding fragment thereof maycomprise a humanised heavy chain variable region which comprises orconsists of the amino acid sequence of any one of SEQ ID NOs: 21 to 23and a humanised light chain variable region which comprises or consistsof the amino acid sequence of any one of SEQ ID NOs: 26 to 28.

It will be appreciated by persons skilled in the art that theabove-defined antibodies or antigen-binding fragments of the inventionmay further comprise a heavy chain constant region, or part thereof.

In one embodiment, the antibody polypeptide comprises a CH1, CH2 and/orCH3 region of an IgG heavy chain (such as an IgG1, IgG2, IgG3 or IgG4heavy chain). Thus, the antibody polypeptide may comprise part or all ofthe constant regions from an IgG1 heavy chain. For example, the antibodypolypeptide may be a Fab fragment comprising CH1 and CL constantregions, combined with any of the above-defined heavy and light variableregions respectively.

Likewise, the above-defined antibodies or antigen-binding fragments ofthe invention may further comprise a light chain constant region, orpart thereof. For example, the antibody polypeptide may comprise a CLregion from a kappa or lambda light chain.

For example, the antibody polypeptide may comprise the followingconstant regions:

-   -   (a) Ig kappa chain C region (Homo sapiens) (UnitProt Accession        No. P01834)

[SEQ ID NO: 29] TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQWKVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

-   -   (b) Ig gamma-1 chain C region (Homo sapiens) (UnitProt Accession        No. P01857)

[SEQ ID NO: 30] ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVSWNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQTYICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGGPSVFLFPPKP KDTLMISRTP EVTCVVVDVS EDPEVKFNWYVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSREEMTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 

In an alternative embodiment, naturally occurring variants of the aboveconstant regions may be utilised (e.g. see Jefferis & Lefranc, 2009,MAbs 1(4):332-8, the disclosures of which are incorporated herein byreference). For example, the light chain constant region may comprise orconsist of SEQ ID NO: 29 having a W40R and/or V83L mutation and/or theheavy china constant region may comprise or consist of SEQ ID NO: 30having a K97R, D239E and/or L241M mutation, or without the C-terminallysine/K (wherein the position of the amino acid mutations is definedusing the Eu Numbering Scheme, which differs from the numbering in SEQID NOS: 29 and 30; see Edelman et al., 1969, Proc. Natl. Acad. Sci. USA,63:78-85, the disclosures of which are incorporated herein byreference).

Thus, exemplary antibody polypeptides of the invention include:

CAN01 and Humanised Versions Thereof:

-   -   (a) a heavy chain comprising a variable region of SEQ ID NO: 1        (or a humanised version thereof, such as SEQ ID NO:8, 9 or 10)        together with a constant region of SEQ ID NO: 30; and    -   (b) a light chain comprising a variable region of SEQ ID NO: 2        (or a humanised version thereof, such as SEQ ID NO:14, 15, 16,        17 or 18) together with a constant region of SEQ ID NO: 29.

CAN03 and Humanised Versions Thereof:

-   -   (a) a heavy chain comprising a variable region of SEQ ID NO: 19        (or a humanised version thereof) together with a constant region        of SEQ ID NO: 30; and    -   (b) a light chain comprising a variable region of SEQ ID NO: 20        (or a humanised version thereof) together with a constant region        of SEQ ID NO: 29.

In a related embodiment, the antibody polypeptide may comprise anantibody Fc-region (e.g. the CH2 and CH3 regions of an IgG heavy chain).It will be appreciated by a skilled person that the Fc portion may befrom an IgG antibody, or from a different class of antibody (such asIgM, IgA, IgD or IgE). In one embodiment, the Fc region is from an IgG1,IgG2, IgG3 or IgG4 antibody.

The Fc region may be naturally-occurring (e.g. part of an endogenouslyproduced antibody) or may be artificial (e.g. comprising one or morepoint mutations relative to a naturally-occurring Fc region and/ormodifications to the carbohydrate moieties within the CH2 domain).

As is well documented in the art, the Fc region of an antibody mediatesits serum half-life and effector functions, such as complement-dependentcytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) andantibody-dependent cell phagocytosis (ADCP).

Engineering the Fc region of a therapeutic monoclonal antibody or Fcfusion protein allows the generation of molecules that are better suitedto the pharmacology activity required of them (Strohl, 2009, Curr OpinBiotechnol 20(6):685-91, the disclosures of which are incorporatedherein by reference).

(a) Engineered Fc Regions for Increased Half-Life

One approach to improve the efficacy of a therapeutic antibody is toincrease its serum persistence, thereby allowing higher circulatinglevels, less frequent administration and reduced doses.

The half-life of an IgG depends on its pH-dependent binding to theneonatal receptor FcRn. FcRn, which is expressed on the surface ofendothelial cells, binds the IgG in a pH-dependent manner and protectsit from degradation.

Some antibodies that selectively bind the FcRn at pH 6.0, but not pH7.4, exhibit a higher half-life in a variety of animal models.

Several mutations located at the interface between the CH2 and CH3domains, such as T250Q/M428L (Hinton et al., 2004, J Biol Chem.279(8):6213-6, the disclosures of which are incorporated herein byreference) and M252Y/S254T/T256E+H433K/N434F (Vaccaro et al., 2005, Nat.Biotechnol. 23(10):1283-8, the disclosures of which are incorporatedherein by reference), have been shown to increase the binding affinityto FcRn and the half-life of IgG1 in vivo.

(b) Engineered Fc Regions for Altered Effector Function

Depending on the therapeutic antibody or Fc fusion protein application,it may be desired to either reduce or increase the effector function(such as ADCC).

For antibodies that target cell-surface molecules, especially those onimmune cells, abrogating effector functions may be required for certainclinical indications.

Conversely, for antibodies intended for oncology use (such as in thetreatment of leukemias and solid tumours; see below), increasingeffector functions may improve the therapeutic activity.

The four human IgG isotypes bind the activating Fcγ receptors (FcγRI,FcγRIIa, FcγRIIIa), the inhibitory FcγRIIb receptor, and the firstcomponent of complement (C1q) with different affinities, yielding verydifferent effector functions (Bruhns et al., 2009, Blood.113(16):3716-25, the disclosures of which are incorporated herein byreference).

Binding of IgG to the FcγRs or C1q depends on residues located in thehinge region and the CH2 domain. Two regions of the CH2 domain arecritical for FcγRs and C1q binding, and have unique sequences in IgG2and IgG4. Substitutions into human IgG1 of IgG2 residues at positions233-236 and IgG4 residues at positions 327, 330 and 331 were shown togreatly reduce ADCC and CDC (Armour et al., 1999, Eur J Immunol.29(8):2613-24; Shields et al., 2001, J Biol Chem. 276(9):6591-604, thedisclosures of which are incorporated herein by reference). Furthermore,Idusogie et al. demonstrated that alanine substitution at differentpositions, including K322, significantly reduced complement activation(Idusogie et al., 2000, J Immunol. 164(8):4178-84, the disclosures ofwhich are incorporated herein by reference). Similarly, mutations in theCH2 domain of murine IgG2A were shown to reduce the binding to FcγRI,and C1q (Steurer. et al., 1995. J Immunol. 155(3):1165-74, thedisclosures of which are incorporated herein by reference).

Numerous mutations have been made in the CH2 domain of human IgG1 andtheir effect on ADCC and CDC tested in vitro (see references citedabove). Notably, alanine substitution at position 333 was reported toincrease both ADCC and CDC (Shields et al., 2001, supra; Steurer et al.,1995, supra). Lazar et al. described a triple mutant (S239D/I332E/A330L)with a higher affinity for FcγRIIIa and a lower affinity for FcγRIIbresulting in enhanced ADCC (Lazar et al., 2006, PNAS 103(11):4005-4010,the disclosures of which are incorporated herein by reference). The samemutations were used to generate an antibody with increased ADCC (Ryan etal., 2007, Mol. Cancer Ther. 6:3009-3018, the disclosures of which areincorporated herein by reference). Richards et al. studied a slightlydifferent triple mutant (S239D/I332E/G236A) with improved FcγRIIIaaffinity and FcγRIIa/FcγRIIb ratio that mediates enhanced phagocytosisof target cells by macrophages (Richards et al., 2008. Mol Cancer Ther.7(8):2517-27, the disclosures of which are incorporated herein byreference).

Due to their lack of effector functions, IgG4 antibodies represent apreferred IgG subclass for receptor blocking without cell depletion(i.e. inhibition of IL-1 signalling). IgG4 molecules can exchangehalf-molecules in a dynamic process termed Fab-arm exchange. Thisphenomenon can also occur in vivo between therapeutic antibodies andendogenous IgG4.

The S228P mutation has been shown to prevent this recombination processallowing the design of less unpredictable therapeutic IgG4 antibodies(Labrijn et al., 2009, Nat Biotechnol. 27(8):767-71, the disclosures ofwhich are incorporated herein by reference).

Examples of engineered Fc regions are shown in Table 1 below.

TABLE 1 Examples of Engineered Fc Effector Isotype Species Mutations*FcR/C1q Binding Function IgG1 Human T250Q/M428L¹ Increased bindingIncreased half- to FcRn life IgG1 Human M252Y/S254T/T256E + Increasedbinding Increased half- H433K/N434F² to FcRn life IgG1 HumanM428L/N434S³ Increased binding Increased half- to FcRn life IgG1 HumanE233P/L234V/L235A/?G236 + Reduced binding ReducedA327G/A330S/P331S^(4,5) to FcγRI ADCC and CDC IgG1 HumanS239D/S298A/I332E + Increased binding Increased S239D/A330L/I332E⁶ toFcγRIIIa ADCC IgG1 Human S239D/I332E⁷ Increased binding Increased toFcγRIIIa ADCC IgG1 Human S298A/E333A/K334A⁸ Increased binding Increasedto FcγRIIIa ADCC IgG1 Human E333A⁹ Increased binding Increased toFcγRIIIa ADCC and CDC IgG1 Human P257I/Q311¹⁰ Increased bindingUnchanged to FcRn half-life IgG1 Human K326W/E333S¹¹ Increased bindingIncreased CDC to C1q IgG1 Human S239D/I332E/G236A¹² Increased IncreasedFcγRIIa/FcγRIIb macrophage ratio phagocytosis IgG1 Human K322A⁸ Reducedbinding Reduced CDC to C1q N297S Reduced (abrogated) ADCC N297Q Reduced(abrogated) ADCC R292P + V305I +/− F243L¹³ Increased ADCC P247I/A339Q¹⁴Increased ADCC IgG4 Human S228P¹⁵ — Reduced Fab- arm exchange IgG2aMouse L235E + E318A/K320A/K322A¹¹ Reduced binding Reduced to FcγRI andC1q ADCC and CDC *The position of the Fc amino acid mutations is definedusing the Eu Numbering Scheme, which differs from the numbering in SEQID NOS: 18 and 19 above; see Edelman et al., 1969, Proc. Natl. Acad.Sci. USA, 63: 78-85) References to Table 1 ¹Hinton et al 2004 J. Biol.Chem. 279(8): 6213-6) ²Vaccaro et al. 2005 Nat Biotechnol. 23(10):1283-8) ³Zalevsky et al 2010 Nat. Biotechnology 28(2): 157-159 ⁴Armour KL. et al., 1999. Eur J Immunol. 29(8): 2613-24 ⁵Shields R L. et al.,2001. J Biol Chem. 276(9): 6591-604 ⁶Masuda et al. 2007, Mol Immunol.44(12): 3122-31 ⁷Bushfield et al 2014, Leukemia 28(11): 2213-21 ⁸Okazakiet al. 2004, J Mol Biol.; 336(5): 1239-49 ⁹Idusogie et al., 2000. JImmunol. 164(8): 4178-84 ¹⁰Datta-Mannan A. et al., 2007. Drug Metab.Dispos. 35: 86-94 ¹¹Steurer W. et al., 1995. J Immunol. 155(3): 1165-74¹²Richards et al. 2008 Mol Cancer There. 7(8): 2517-27 ¹³U.S. Pat. No.7,960,512 B2 ¹⁴EP 2 213 683 ¹⁵Labrijn A F. et al., 2009. Nat Biotechnol.27(8): 767-71

In a further embodiment, the effector function of the Fc region may bealtered through modification of the carbohydrate moieties within the CH2domain therein.

For example, it is known that therapeutic antibodies lacking or low infucose residues in the Fc region may exhibit enhanced ADCC activity inhumans (for example, see Peipp et al., 2008, Blood 112(6):2390-9,Yamane-Ohnuki & Satoh, 2009, MAbs 1(3):230-26, Iida et al., 2009, BMCCancer 9; 58 (the disclosures of which are incorporated herein byreference). Low fucose antibody polypeptides may be produced byexpression in cells cultured in a medium containing an inhibitor ofmannosidase, such as kinfunensine (see Example I below).

Other methods to modify glycosylation of an antibody into a low fucoseformat include the use of the bacterial enzymeGDP-6-deoxy-D-lyxo-4-hexulose reductase in cells not able to metaboliserhamnose (e.g. using the GlymaxX® technology of ProBioGen AG, Berlin,Germany).

Another method to create low fucose antibodies is by inhibition ordepletion of alpha-(1,6)-fucosyltransferase in the antibody-producingcells (e.g. using the Potelligent® CHOK1SV technology of Lonza Ltd,Basel, Switzerland).

As noted above, the antibody polypeptides of the invention may exert aninhibitory action on IL-1 signalling (see Examples E, L and M), eitherin addition to or in the absence of any Fc-mediated effector functions.

In one embodiment, the antibody polypeptides of the invention may exertan inhibitory action on one or more additional (or alternative)cytokines within the IL-1 superfamily, including but not limited toIL-33 and/or IL-36.

Interleukin-33 (IL-33) induces helper T cells, mast cells, eosinophilsand basophils to produce type 2 cytokines. This cytokine was previouslynamed NF-HEV ‘nuclear factor (NF) in high endothelial venules’ (HEVs)since it was originally identified in these specialized cells. IL-33mediates its biological effects by interacting with the receptors ST2(also known as IL1RL1) and IL-1 Receptor Accessory Protein (IL1RAP),activating intracellular molecules in the NF-κB and MAP kinasesignalling pathways that drive production of type 2 cytokines (e.g. IL-5and IL-13) from polarised Th2 cells. The induction of type 2 cytokinesby IL-33 in vivo is believed to induce the severe pathological changesobserved in mucosal organs following administration of IL-33.

Interleukin-36 (IL-36) is a cytokine that predominantly acts on naiveCD4+ T cells via the IL-36 receptor. It is known to activate NF-κB andmitogen-activated protein kinases to play a role in skin pathology. Ithas also been found to activate T cell proliferation and release ofIL-2.

It will be appreciated by persons skilled in the art that the antibodypolypeptide of the invention may inhibit IL-1, IL-33 and/or IL-36signalling in whole or in part. For example, signalling may be inhibitedby at least 10%, 20%, 30%, 50%, 75% or more relative to signalling inthe absence of the polypeptide of the invention.

The degree of inhibition of IL-1, IL-33 and/or IL-36 signalling by thepolypeptide of the invention may be determined using methods well knownin the art.

For example, inhibition of IL-1 signalling may be measured as describedin Examples E, L and M below.

Likewise, inhibition of IL-33 signalling may be measured as described inExample E, L and M.

Inhibition of IL-36 signalling may be measured by methods known in theart. For example, IL-36 stimulation of synovial fibroblasts leads toNF-κB and MAP kinase activation. Alternatively, IL-36-α, -β and -γincrease T-cell proliferation in response to antiCD3/anti-CD28stimulation (see Vigne et al., 2012, Blood 120(17):3478-87, thedisclosures of which are incorporated herein by reference).

In one embodiment, the antibody or antigen-binding fragment thereof mayfurther comprise a moiety for increasing the in vivo half-life of theantibody or antigen-binding fragment, such as but not limited topolyethylene glycol (PEG), human serum albumin, glycosylation groups,fatty acids and dextran. Such further moieties may be conjugated orotherwise combined with the binding moiety using methods well known inthe art.

It will be appreciated by persons skilled in the art that the antibodypolypeptides of the invention may comprise or consist of one or moreamino acids which have been modified or derivatised.

Chemical derivatives of one or more amino acids may be achieved byreaction with a functional side group. Such derivatised moleculesinclude, for example, those molecules in which free amino groups havebeen derivatised to form amine hydrochlorides, p-toluene sulphonylgroups, carboxybenzoxy groups, t-butyloxycarbonyl groups, chloroacetylgroups or formyl groups. Free carboxyl groups may be derivatised to formsalts, methyl and ethyl esters or other types of esters and hydrazides.Free hydroxyl groups may be derivatised to form O-acyl or O-alkylderivatives. Also included as chemical derivatives are those peptideswhich contain naturally occurring amino acid derivatives of the twentystandard amino acids. For example: 4-hydroxyproline may be substitutedfor proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine and ornithine for lysine. Derivatives alsoinclude peptides containing one or more additions or deletions as longas the requisite activity is maintained. Other included modificationsare amidation, amino terminal acylation (e.g. acetylation orthioglycolic acid amidation), terminal carboxylamidation (e.g. withammonia or methylamine), and the like terminal modifications.

It will be further appreciated by persons skilled in the art thatpeptidomimetic compounds may also be useful. The term ‘peptidomimetic’refers to a compound that mimics the conformation and desirable featuresof a particular peptide as a therapeutic agent.

For example, the said polypeptide includes not only molecules in whichamino acid residues are joined by peptide (—CO—NH—) linkages but alsomolecules in which the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al. (1997) J. Immunol. 159,3230-3237, which is incorporated herein by reference. This approachinvolves making pseudo-peptides containing changes involving thebackbone, and not the orientation of side chains. Retro-inversepeptides, which contain NH—CO bonds instead of CO—NH peptide bonds, aremuch more resistant to proteolysis. Alternatively, the said polypeptidemay be a peptidomimetic compound wherein one or more of the amino acidresidues are linked by a -y(CH₂NH)— bond in place of the conventionalamide linkage.

In a further alternative, the peptide bond may be dispensed withaltogether provided that an appropriate linker moiety which retains thespacing between the carbon atoms of the amino acid residues is used; itmay be advantageous for the linker moiety to have substantially the samecharge distribution and substantially the same planarity as a peptidebond.

It will also be appreciated that the said polypeptide may convenientlybe blocked at its N- or C-terminus so as to help reduce susceptibilityto exo-proteolytic digestion.

A variety of un-coded or modified amino acids such as D-amino acids andN-methyl amino acids have also been used to modify mammalian peptides.In addition, a presumed bioactive conformation may be stabilised by acovalent modification, such as cyclisation or by incorporation of lactamor other types of bridges, for example see Veber et al., 1978, Proc.Natl. Acad. Sci. USA 75:2636 and Thursell et al., 1983, Biochem.Biophys. Res. Comm. 111:166, which are incorporated herein by reference.

The antibody polypeptides of the invention may be augmented with afunctional moiety to facilitate their intended use, for example as adiagnostic (e.g. in vivo imaging) agent or therapeutic agent. Thus, inone embodiment, the antibody polypeptide is linked, directly orindirectly, to a therapeutic moiety.

In one embodiment, the antibody or antigen-binding fragment thereofaccording to any one of the preceding claim further comprising atherapeutic (e.g. cytotoxic) moiety.

Any suitable therapeutic moiety may be used. A suitable therapeuticmoiety is one that is capable of reducing or inhibiting the growth, orin particular killing, a cancer cell (or associated stem cells orprogenitor cells). For example, the therapeutic agent may be a cytotoxicmoiety. The cytotoxic moiety may comprise or consist of one or moreradioisotopes. For example, the one or more radioisotopes may each beindependently selected from the group consisting of beta-emitters,Auger-emitters, conversion electron-emitters, alpha-emitters, and lowphoton energy-emitters. It may be desired that the one or moreradioisotopes each independently has an emission pattern of locallyabsorbed energy that creates a high absorbed dose in the vicinity of theagent. Exemplary radioisotopes may include long-range beta-emitters,such as ⁹⁰Y, ³²P, ¹⁸⁶Re/¹⁸⁸Re; ¹⁶⁶Ho, ⁷⁶As/⁷⁷As, ⁸⁹Sr, ¹⁵³Sm; mediumrange beta-emitters, such as ¹³¹I, ¹⁷⁷Lu, ⁶⁷Cu, ¹⁶¹Tb, ¹⁰⁵Rh; low-energybeta-emitters, such as ⁴⁵Ca or ³⁵S; conversion or Auger-emitters, suchas ⁵¹Cr, ⁶⁷Ga, ⁹⁹Tc^(m), ¹¹¹In, ^(114m)In, ¹²³I, ¹²⁵I, ²⁰¹Tl; andalpha-emitters, such as ²¹²Bi, ²¹³Bi, ²²³Ac, ²²⁵Ac, ²¹²Pb, ²⁵⁵Fm, ²²³Ra,¹⁴⁹Tb and ²²¹At. Other radionuclides are available and will be possibleto use for therapy.

In one preferred embodiment, the antibody polypeptide is linked to (orotherwise labelled with) the radioisotope ¹⁷⁷Lu.

Alternatively, the therapeutic moiety may comprise or consist of one ormore therapeutic (such as cytotoxic) drugs, for example, a cytostaticdrug; an anti-androgen drug; cortisone and derivatives thereof; aphosphonate; a testosterone-5-α-reductase inhibitor; a boron addend; acytokine; thapsigargin and its metabolites; a toxin (such as saporin orcalicheamicin); a chemotherapeutic agent (such as an antimetabolite); orany other therapeutic or cytotoxic drug useful in the treatment ofcancers.

Exemplary therapeutic/cytotoxic drugs may, for example, include:

-   -   Cytostatics, in particular those with dose-limiting        side-effects, including but not limited to cyclophosamide,        chlorambucil, ifosfamide, busulphane, lomustine, taxanes,        estramustine phosphate and other nitrogen mustards, antibiotics        (including doxorubicine, calicheamicines and esperamicine),        vinca alkaloids, azaridines, platinum-containing compounds,        endostatin, alkyl sulfonates, nitrosoureas, triazenes, folic        acid analoges, pyrimidine analoges, purine analogs, enzymes,        substituted urea, methyl-hydrazine derivatives, daunorubicin,        amphipathic amines,    -   Anti-androgens such as flutamide and bikalutamide and        metabolites thereof;    -   Cortisone and derivatives thereof;    -   Phosphonates such as diphophonate and buphosphonate;    -   Testosterone-5-α-reductase inhibitors;    -   Boron addends;    -   Cytokines;    -   Thapsigargin and its metabolites.    -   Bacterial toxins or engineered variants of these.    -   Immune modulators

Alternatively, the cytotoxic moiety may comprise or consist of one ormore moieties suitable for use in activation therapy, such as photonactivation therapy, neutron activation therapy, neutron-induced Augerelectron therapy, synchrotron irradiation therapy or low energy X-rayphoton activation therapy.

For example, with the antibody polypeptides of the invention there willbe the potential of using synchrotron radiation (or low energy X-rays)for the advancement of radiotherapy, primarily focusing on so calledphoto-activation radiotherapy (PAT), in which the local energydeposition from external X-ray irradiation is enhanced in the cancertissue by the interaction with a pre-administered, high-Ztumour-targeting agent.

The PAT treatment modality utilises monochromatic X-rays from asynchrotron source, such as provided by the ID17 biomedical beamline atthe European Synchrotron Radiation Facility (ESRF) in Grenoble, and asanticipated to be available at other facilities in the future such asthe new Swedish synchrotron facility, Max-IV.

Research on “induced Auger electron tumour therapy”, to be conducted atthe coming European Spallation Source (ESS) in Lund, provides a furtherpotential treatment modality. Reactor-produced thermal and semi-thermalneutrons have for long been used for Boron-Neutron-Capture-Therapy,BNCT, both for pre-clinical experiments and for treatment of braintumours with the induced alpha-particles and the recoil nucleus (⁷L)that give a high locally absorbed energy. A similar approach is to useneutrons and suitable tumour-targeting molecules labelled with stablenuclei with high cross-section for neutrons. Antibodies or peptides canfor instance be labelled with stable Gadolinium (¹⁵⁷Gd) and act as thetarget molecule for the neutrons that are captured by the Gd-nucleus, socalled Gadolinium Neutron Capture Therapy (GdNCT). By Monte Carlotechniques, the dose distribution in the tumour and the surroundingtissues is calculated as it results from γ-photons, neutrons, nuclearrecoils, as well as characteristic x-rays, internal conversion andAuger-electrons from gadolinium or other potential elements.

Optionally, the antibody polypeptide of the invention may furthercomprise a detectable moiety. For example, a detectable moiety maycomprise or consist of a radioisotope, such as a radioisotope selectedfrom the group consisting of ^(99m)Tc, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr,¹²³I and ²⁰¹Tl Optionally, the agent may comprise a pair of detectableand cytotoxic radionuclides, such as ⁸⁶Y/⁹⁰Y or ¹²⁴I/²¹¹At.Alternatively, the antibody polypeptide may comprise a radioisotope thatis capable of simultaneously acting in a multi-modal manner as adetectable moiety and also as a cytotoxic moiety to provide so-called“Multimodality theragnostics”. The binding moieties may thus be coupledto nanoparticles that have the capability of multi-imaging (for example,SPECT, PET, MRI, Optical, or Ultrasound) together with therapeuticcapability using cytotoxic drugs, such as radionuclides or chemotherapyagents.

Alternatively, the detectable moiety may comprise or consist of aparamagnetic isotope, such as a paramagnetic isotope is selected fromthe group consisting of ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr and ⁵⁶Fe.

In the case that the antibody polypeptide comprises a detectable moiety,then the detectable moiety may be detectable by an imaging techniquesuch as SPECT, PET, MRI, optical or ultrasound imaging.

Therapeutic and/or detectable moieties (such as a radioisotope,cytotoxic moiety or the like) may be linked directly, or indirectly, tothe antibody or fragment thereof. Suitable linkers are known in the artand include, for example, prosthetic groups, non-phenolic linkers(derivatives of N-succimidyl-benzoates; dodecaborate), chelatingmoieties of both macrocyclics and acyclic chelators, such as derivativesof 1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA),deferoxamine (DFO), derivatives of diethylenetriaminepentaacetic avid(DTPA), derivatives ofS-2-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triaceticacid (NOTA) and derivatives of1,4,8,11-tetraazacyclodocedan-1,4,8,11-tetraacetic acid (TETA),derivatives of 3,6,9,15-Tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-4-(S)-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid (PCTA),derivatives of5-S-(4-Aminobenzyl)-1-oxa-4,7,10-triazacyclododecane-4,7,10-tris(aceticacid) (DO3A) and other chelating moieties.

One preferred linker is DTPA, for example as used in¹⁷⁷Lu-DTPA-[antibody polypeptide of the invention]. A further preferredlinker is deferoxamine, DFO, for example as used in ⁸⁹Zr-DFO-[antibodypolypeptide of the invention].

As discussed above, methods for the production of antibody polypeptidesof the invention are well known in the art.

Conveniently, the antibody polypeptide is or comprises a recombinantpolypeptide. Suitable methods for the production of such recombinantpolypeptides are well known in the art, such as expression inprokaryotic or eukaryotic hosts cells (for example, see Green &Sambrook, 2012, Molecular Cloning, A Laboratory Manual, Fourth Edition,Cold Spring Harbor, N.Y., the relevant disclosures in which document arehereby incorporated by reference).

Antibody polypeptides of the invention can also be produced using acommercially available in vitro translation system, such as rabbitreticulocyte lysate or wheatgerm lysate (available from Promega).Preferably, the translation system is rabbit reticulocyte lysate.Conveniently, the translation system may be coupled to a transcriptionsystem, such as the TNT transcription-translation system (Promega). Thissystem has the advantage of producing suitable mRNA transcript from anencoding DNA polynucleotide in the same reaction as the translation.

It will be appreciated by persons skilled in the art that antibodypolypeptides of the invention may alternatively be synthesisedartificially, for example using well known liquid-phase or solid phasesynthesis techniques (such as t-Boc or Fmoc solid-phase peptidesynthesis).

A second aspect of the invention provides an isolated nucleic acidmolecule encoding an antibody or antigen-binding fragment of the firstaspect of the invention, or a component polypeptide chain thereof. By“nucleic acid molecule” we include DNA (e.g. genomic DNA orcomplementary DNA) and mRNA molecules, which may be single- ordouble-stranded. By “isolated” we mean that the nucleic acid molecule isnot located or otherwise provided within a cell.

In one embodiment, the nucleic acid molecule is a cDNA molecule.

It will be appreciated by persons skilled in the art that the nucleicacid molecule may be codon-optimised for expression of the antibodypolypeptide in a particular host cell, e.g. for expression in humancells (for example, see Angov, 2011, Biotechnol. J. 6(6):650-659, thedisclosures of which are incorporated herein by reference).

Also included within the scope of the invention are the following:

-   (a) a third aspect of the invention provides a vector (such as an    expression vector) comprising a nucleic acid molecule according to    the second aspect of the invention;-   (b) a fourth aspect of the invention provides a host cell (such as a    mammalian cell, e.g. human cell, or Chinese hamster ovary cell, e.g.    CHOK1SV cells) comprising a nucleic acid molecule according to the    second aspect of the invention or a vector according to the third    aspect of the invention; and-   (c) a fifth aspect of the invention provides a method of making an    antibody polypeptide according to the first aspect of the invention    comprising culturing a population of host cells according to the    fourth aspect of the invention under conditions in which said    polypeptide is expressed, and isolating the polypeptide therefrom.

A sixth aspect of the invention provides a pharmaceutical compositioncomprising a pharmaceutically effective amount of an antibody orantigen-binding fragment according to the first aspect of the inventionand a pharmaceutically-acceptable diluent, carrier, adjuvant orexcipient.

It will be appreciated by persons skilled in the art that additionalcompounds may also be included in the pharmaceutical compositions,including, chelating agents such as EDTA, citrate, EGTA or glutathione.

The pharmaceutical compositions may be prepared in a manner known in theart that is sufficiently storage stable and suitable for administrationto humans and animals. For example, the pharmaceutical compositions maybe lyophilised, e.g. through freeze drying, spray drying, spray cooling,or through use of particle formation from supercritical particleformation.

By “pharmaceutically acceptable” we mean a non-toxic material that doesnot decrease the effectiveness of the IL1RAP-binding activity of theantibody polypeptide of the invention. Such pharmaceutically acceptablebuffers, carriers or excipients are well-known in the art (seeRemington's Pharmaceutical Sciences, 18th edition, A. R Gennaro, Ed.,Mack Publishing Company (1990) and handbook of PharmaceuticalExcipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000), thedisclosures of which are incorporated herein by reference).

The term “buffer” is intended to mean an aqueous solution containing anacid-base mixture with the purpose of stabilising pH. Examples ofbuffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes,HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate,borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate,CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole,imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO andTES.

The term “diluent” is intended to mean an aqueous or non-aqueoussolution with the purpose of diluting the antibody polypeptide in thepharmaceutical preparation. The diluent may be one or more of saline,water, polyethylene glycol, propylene glycol, ethanol or oils (such assafflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

The term “adjuvant” is intended to mean any compound added to theformulation to increase the biological effect of the antibodypolypeptide of the invention. The adjuvant may be one or more of zinc,copper or silver salts with different anions, for example, but notlimited to fluoride, chloride, bromide, iodide, tiocyanate, sulfite,hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate,tartrate, and acetates of different acyl composition. The adjuvant mayalso be cationic polymers such as cationic cellulose ethers, cationiccellulose esters, deacetylated hyaluronic acid, chitosan, cationicdendrimers, cationic synthetic polymers such as poly(vinyl imidazole),and cationic polypeptides such as polyhistidine, polylysine,polyarginine, and peptides containing these amino acids.

The excipient may be one or more of carbohydrates, polymers, lipids andminerals. Examples of carbohydrates include lactose, glucose, sucrose,mannitol, and cyclodextrines, which are added to the composition, e.g.for facilitating lyophilisation. Examples of polymers are starch,cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose,alginates, carageenans, hyaluronic acid and derivatives thereof,polyacrylic acid, polysulphonate, polyethylenglycol/polyethylene oxide,polyethyleneoxide/polypropylene oxide copolymers,polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, andpolyvinylpyrrolidone, all of different molecular weight, which are addedto the composition, e.g., for viscosity control, for achievingbioadhesion, or for protecting the lipid from chemical and proteolyticdegradation. Examples of lipids are fatty acids, phospholipids, mono-,di-, and triglycerides, ceramides, sphingolipids and glycolipids, all ofdifferent acyl chain length and saturation, egg lecithin, soy lecithin,hydrogenated egg and soy lecithin, which are added to the compositionfor reasons similar to those for polymers. Examples of minerals aretalc, magnesium oxide, zinc oxide and titanium oxide, which are added tothe composition to obtain benefits such as reduction of liquidaccumulation or advantageous pigment properties.

The antibody polypeptides of the invention may be formulated into anytype of pharmaceutical composition known in the art to be suitable forthe delivery thereof.

In one embodiment, the pharmaceutical compositions of the invention maybe in the form of a liposome, in which the antibody polypeptide iscombined, in addition to other pharmaceutically acceptable carriers,with amphipathic agents such as lipids, which exist in aggregated formsas micelles, insoluble monolayers and liquid crystals. Suitable lipidsfor liposomal formulation include, without limitation, monoglycerides,diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bileacids, and the like. Suitable lipids also include the lipids abovemodified by poly(ethylene glycol) in the polar headgroup for prolongingbloodstream circulation time. Preparation of such liposomal formulationsis can be found in for example U.S. Pat. No. 4,235,871, the disclosuresof which are incorporated herein by reference.

The pharmaceutical compositions of the invention may also be in the formof biodegradable microspheres. Aliphatic polyesters, such as poly(lacticacid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA)or poly(caprolactone) (PCL), and polyanhydrides have been widely used asbiodegradable polymers in the production of microspheres. Preparationsof such microspheres can be found in U.S. Pat. No. 5,851,451 and in EP 0213 303, the disclosures of which are incorporated herein by reference.

In a further embodiment, the pharmaceutical compositions of theinvention are provided in the form of polymer gels, where polymers suchas starch, cellulose ethers, cellulose carboxymethylcellulose,hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethylcellulose, alginates, carageenans, hyaluronic acid and derivativesthereof, polyacrylic acid, polyvinyl imidazole, polysulphonate,polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropyleneoxide copolymers, polyvinylalcohol/polyvinylacetate of different degreeof hydrolysis, and polyvinylpyrrolidone are used for thickening of thesolution containing the agent. The polymers may also comprise gelatin orcollagen.

Alternatively, the antibody polypeptide may simply be dissolved insaline, water, polyethylene glycol, propylene glycol, ethanol or oils(such as safflower oil, corn oil, peanut oil, cottonseed oil or sesameoil), tragacanth gum, and/or various buffers.

It will be appreciated that the pharmaceutical compositions of theinvention may include ions and a defined pH for potentiation of actionof the active antibody polypeptide. Additionally, the compositions maybe subjected to conventional pharmaceutical operations such assterilisation and/or may contain conventional adjuvants such aspreservatives, stabilisers, wetting agents, emulsifiers, buffers,fillers, etc.

The pharmaceutical compositions according to the invention may beadministered via any suitable route known to those skilled in the art.Thus, possible routes of administration include parenteral (intravenous,subcutaneous, and intramuscular), topical, ocular, nasal, pulmonar,buccal, oral, parenteral, vaginal and rectal. Also administration fromimplants is possible.

In one preferred embodiment, the pharmaceutical compositions areadministered parenterally, for example, intravenously,intracerebroventricularly, intraarticularly, intra-arterially,intraperitoneally, intrathecally, intraventricularly, intrasternally,intracranially, intramuscularly or subcutaneously, or they may beadministered by infusion techniques. They are conveniently used in theform of a sterile aqueous solution which may contain other substances,for example, enough salts or glucose to make the solution isotonic withblood. The aqueous solutions should be suitably buffered (preferably toa pH of from 3 to 9), if necessary. The preparation of suitableparenteral formulations under sterile conditions is readily accomplishedby standard pharmaceutical techniques well known to those skilled in theart.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Thus, the pharmaceutical compositions of the invention are particularlysuitable for parenteral, e.g. intravenous, administration.

Alternatively, the pharmaceutical compositions may be administeredintranasally or by inhalation (for example, in the form of an aerosolspray presentation from a pressurised container, pump, spray ornebuliser with the use of a suitable propellant, such asdichlorodifluoromethane, trichlorofluoro-methane,dichlorotetrafluoro-ethane, a hydrofluoroalkane such as1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane(HFA 227EA3), carbon dioxide or other suitable gas). In the case of apressurised aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. The pressurised container, pump,spray or nebuliser may contain a solution or suspension of the activepolypeptide, e.g. using a mixture of ethanol and the propellant as thesolvent, which may additionally contain a lubricant, e.g. sorbitantrioleate. Capsules and cartridges (made, for example, from gelatin) foruse in an inhaler or insufflator may be formulated to contain a powdermix of a compound of the invention and a suitable powder base such aslactose or starch.

The pharmaceutical compositions will be administered to a patient in apharmaceutically effective dose. A ‘therapeutically effective amount’,or ‘effective amount’, or ‘therapeutically effective’, as used herein,refers to that amount which provides a therapeutic effect for a givencondition and administration regimen. This is a predetermined quantityof active material calculated to produce a desired therapeutic effect inassociation with the required additive and diluent, i.e. a carrier oradministration vehicle. Further, it is intended to mean an amountsufficient to reduce and most preferably prevent, a clinicallysignificant deficit in the activity, function and response of the host.Alternatively, a therapeutically effective amount is sufficient to causean improvement in a clinically significant condition in a host. As isappreciated by those skilled in the art, the amount of a compound mayvary depending on its specific activity. Suitable dosage amounts maycontain a predetermined quantity of active composition calculated toproduce the desired therapeutic effect in association with the requireddiluent. In the methods and use for manufacture of compositions of theinvention, a therapeutically effective amount of the active component isprovided. A therapeutically effective amount can be determined by theordinary skilled medical or veterinary worker based on patientcharacteristics, such as age, weight, sex, condition, complications,other diseases, etc., as is well known in the art. The administration ofthe pharmaceutically effective dose can be carried out both by singleadministration in the form of an individual dose unit or else severalsmaller dose units and also by multiple administrations of subdivideddoses at specific intervals. Alternatively, the does may be provided asa continuous infusion over a prolonged period.

In the context of diagnostic use of the antibody polypeptides of theinvention, a ‘pharmaceutically effective amount’, or ‘effective amount’,or ‘diagnostically effective’, as used herein, refers to that amountwhich provides a detectable signal for diagnosis, e.g. for in vivoimaging purposes.

The antibody polypeptides can be formulated at various concentrations,depending on the efficacy/toxicity of the polypeptide being used. Forexample, the formulation may comprise the active antibody polypeptide ata concentration of between 0.1 μM and 1 mM, more preferably between 1 μMand 500 μM, between 500 μM and 1 mM, between 300 μM and 700 μM, between1 μM and 100 μM, between 100 μM and 200 μM, between 200 μM and 300 μM,between 300 μM and 400 μM, between 400 μM and 500 μM, between 500 μM and600 μM, between 600 μM and 700 μM, between 800 μM and 900 μM or between900 μM and 1 mM. Typically, the formulation comprises the activeantibody polypeptide at a concentration of between 300 μM and 700 μM.

Typically, the therapeutic dose of the antibody polypeptide (with orwithout a therapeutic moiety) in a human patient will be in the range of100 μg to 700 mg per administration (based on a body weight of 70 kg).For example, the maximum therapeutic dose may be in the range of 0.1 to10 mg/kg per administration, e.g. between 0.1 and 5 mg/kg or between 1and 5 mg/kg or between 0.1 and 2 mg/kg. It will be appreciated that sucha dose may be administered at different intervals, as determined by theoncologist/physician; for example, a dose may be administered daily,twice-weekly, weekly, bi-weekly or monthly.

It will be appreciated by persons skilled in the art that thepharmaceutical compositions of the invention may be administered aloneor in combination with other therapeutic agents used in the treatment ofcancers, such as antimetabolites, alkylating agents, anthracyclines andother cytotoxic antibiotics, vinca alkyloids, etoposide, platinumcompounds, taxanes, topoisomerase I inhibitors, other cytostatic drugs,antiproliferative immunosuppressants, corticosteroids, sex hormones andhormone antagonists, and other therapeutic antibodies (such astrastuzumab).

A seventh aspect of the invention provides an antibody orantigen-binding fragment thereof according to the first aspect of theinvention for use in medicine.

A related eighth aspect of the invention provides an antibody orantigen-binding fragment thereof according to the first aspect of theinvention for use in inducing cell death and/or inhibiting the growthand/or proliferation of pathological cells associated with a neoplasticdisorder in a subject, or stem cells or progenitor cells thereof,wherein the cells express IL1RAP.

A further related ninth aspect of the invention provides an antibody orantigen-binding fragment according to the first aspect of the inventionfor use in the treatment and/or diagnosis of a neoplastic disorder in asubject, wherein the neoplastic disorder is associated with cellsexpressing IL1RAP.

By ‘treatment’ we include both therapeutic and prophylactic treatment ofthe patient. The term ‘prophylactic’ is used to encompass the use of anagent, or formulation thereof, as described herein which either preventsor reduces the likelihood of a neoplastic disorder, or the spread,dissemination, or metastasis of cancer cells in a patient or subject.The term ‘prophylactic’ also encompasses the use of an agent, orformulation thereof, as described herein to prevent recurrence of aneoplastic disorder in a patient who has previously been treated for theneoplastic disorder.

By “diagnosis” we include the detection of cancerous cells, either invivo (i.e. within the body of a patient) or ex vivo (i.e. within atissue or cell sample removed from the body of a patient).

By “a neoplastic disorder associated with cells expressing IL1RAP” weinclude such disorders wherein the pathological cells which areresponsible, directly or indirectly, for the disorder express IL1 RAP onthe cell surface. It will be appreciated that the cells expressingIL1RAP may be cancer cells, e.g. tumour cells, per se. In addition, suchcells include pathological stem cells (i.e. cancer stem cells, or CSCs)and progenitor cells which are responsible, directly or indirectly, forthe development of a neoplastic disorder in an individual. Examples ofCSCs are disclosed in Visvader & Lindeman, 2008, Nat Rev Cancer8:755-768, the disclosures of which are incorporated herein byreference.

Alternatively, or in addition, the cells expressing IL1RAP may beassociated indirectly with the neoplastic disorder, for example, theymay mediate cellular processes required for the neoplastic cells tosurvive. The antibody agent of the invention may in this event targetcells essential for the blood supply of the tumour (angiogenesis), thetumour stroma or cells inhibiting a beneficial immune response directedagainst the malignant cells (e.g. suppressive macrophages or T-cells).

Depending upon whether it is therapeutically desirable to kill thetarget cells expressing IL1RAP, an antibody or antigen-binding fragmentaccording to the first aspect of the invention mau be used that itcapable of inducing ADCC. For example, where the target cells IL1RAP arecancer cells (such as CML, AML, ALL, melanoma, lung cancer cells, etc)it may be advantageous for the antibody or antigen-binding fragment tobe capable of inducing ADCC in order to eliminate such cells. However,it will be appreciated that a therapeutic benefit may also be achievedusing an antibody or antigen-binding fragment that lacks ADCC activity,for example through inhibition of IL-1 (or IL-33) signalling leading toreduced angiogenesis in the vicinity of a tumour.

In one embodiment, the neoplastic disorder is a neoplastic haematologicdisorder.

For example, the antibody or antigen-binding fragment thereof may be foruse in the treatment and/or diagnosis of a neoplastic disorder selectedfrom the group consisting of chronic myeloid leukemia (CML),myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS),acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

In a further embodiment, the antibody or antigen-binding fragmentthereof is for use in the treatment and/or diagnosis of a neoplasticdisorder associated with the formation of solid tumours within thesubject's body.

Thus, the antibody or antigen-binding fragment thereof may be for use inthe treatment of a neoplastic disorder selected from the groupconsisting of prostate cancer, breast cancer, lung cancer, colorectalcancer, melanomas, bladder cancer, brain/CNS cancer, cervical cancer,oesophageal cancer, gastric cancer, head/neck cancer, kidney cancer,liver cancer, lymphomas, ovarian cancer, pancreatic cancer, andsarcomas.

In relation to the therapeutic and prophylactic aspects of theinvention, it will be appreciated by persons skilled in the art thatbinding of the antibody polypeptide to IL1RAP present on the surface ofthe cells associated with the neoplastic disorder may lead to amodulation (i.e. an increase or decrease) of a biological activity ofIL1RAP. However, such a modulatory effect is not essential; for example,the antibody polypeptides of the invention may elicit a therapeutic andprophylactic effect simply by virtue of binding to IL1RAP on the surfaceof the cells associated with the solid tumour, which in turn may triggerthe immune system to induce cell death (e.g. by ADCC and/or by thepresence within the agent of a cytotoxic/radioactive moiety).

By “biological activity of IL1RAP” we include any interaction orsignalling event which involves IL1RAP on the cells associated with theneoplastic disorder. For example, in one embodiment the antibodypolypeptide is capable of blocking binding of one or more co-receptorsto IL1RAP (such as IL1R1, ST2, C-KIT and/or IL1RL2).

Such inhibition of the biological activity of IL1RAP by an antibodypolypeptide of the invention may be in whole or in part. For example,the agent may inhibit the biological activity of IL1RAP by at least 10%,preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and mostpreferably by 100% compared to the biological activity of IL1RAP incells associated with the neoplastic disorder which have not beenexposed to the antibody polypeptide. In a preferred embodiment, theantibody polypeptide is capable of inhibiting the biological activity ofIL1RAP by 50% or more compared to the biological activity of IL1RAP incells associated with the neoplastic disorder which have not beenexposed to the antibody polypeptide.

Likewise, it will be appreciated that inhibition of growth and/orproliferation of the cells associated with the neoplastic disorder maybe in whole or in part. For example, the antibody polypeptide mayinhibit the growth and/or proliferation of the cells associated with theneoplastic disorder by at least 10%, preferably at least 20%, 30%, 40%,50%, 60%, 70%, 80% or 90%, and most preferably by 100% compared to thegrowth and/or proliferation of cells associated with the neoplasticdisorder which have not been exposed to the antibody polypeptide.

A tenth aspect of the invention provides use of an antibody orantigen-binding fragment thereof according to the first aspect of theinvention in the preparation of a medicament for the treatment of aneoplastic disorder in a subject, wherein the neoplastic disorder isassociated with cells expressing IL1RAP.

In a related aspect, the invention provides use of an antibody orantigen-binding fragment thereof according to the first aspect of theinvention in the preparation of an agent for the diagnosis and/orprognosis of a neoplastic disorder in a subject, wherein the neoplasticdisorder is associated with cells expressing IL1RAP. Thus, there isprovided a diagnostic and/or prognostic agent for use in detecting cells(expressing IL1RAP) that are associated with a neoplastic disorder. Theinvention further provides the use of an IL1RAP-specific antibody orantigen-binding fragment thereof according to the first aspect of theinvention in the preparation of a kit for diagnosing and/or prognosing aneoplastic disorder in a subject.

In one embodiment, the neoplastic disorder is a neoplastic haematologicdisorder.

For example, the antibody or antigen-binding fragment thereof may be foruse in the treatment and/or diagnosis of a neoplastic disorder selectedfrom the group consisting of chronic myeloid leukemia (CML),myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS),acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

In a further embodiment, the antibody or antigen-binding fragmentthereof is for use in the treatment and/or diagnosis of a neoplasticdisorder associated with the formation of solid tumours within thesubject's body.

Thus, the antibody or antigen-binding fragment thereof may be for use inthe treatment and/or diagnosis of a neoplastic disorder selected fromthe group consisting of prostate cancer, breast cancer, lung cancer,colorectal cancer, melanomas, bladder cancer, brain/CNS cancer, cervicalcancer, oesophageal cancer, gastric cancer, head/neck cancer, kidneycancer, liver cancer, lymphomas, ovarian cancer, pancreatic cancer, andsarcomas.

An eleventh aspect of the invention provides a method for the treatmentor diagnosis of a neoplastic disorder in a subject, comprising the stepof administering to the subject an effective amount of an antibody orantigen-binding fragment thereof according to the first aspect of theinvention, wherein the neoplastic disorder is associated with cellsexpressing IL1RAP.

In one embodiment, the neoplastic disorder is a neoplastic haematologicdisorder.

For example, the method may be for use in the treatment and/or diagnosisof a neoplastic disorder selected from the group consisting of chronicmyeloid leukemia (CML), myeloproliferative disorders (MPD),myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) andacute myeloid leukemia (AML).

In a further embodiment, the method is for use in the treatment and/ordiagnosis of a neoplastic disorder associated with the formation ofsolid tumours within the subject's body.

Thus, the method may be for use in the treatment of a neoplasticdisorder selected from the group consisting of prostate cancer, breastcancer, lung cancer, colorectal cancer, melanomas, bladder cancer,brain/CNS cancer, cervical cancer, oesophageal cancer, gastric cancer,head/neck cancer, kidney cancer, liver cancer, lymphomas, ovariancancer, pancreatic cancer, and sarcomas.

In a further embodiment, the antibody polypeptides and formulations ofthe invention may be used to treat patients or subjects who suffer fromor are at risk of suffering a disease or condition susceptible totreatment with an inhibitor of IL-1 (or IL-33) signalling.

Thus, a twelfth aspect of the invention provides an antibody orantigen-binding fragment thereof according to the first aspect of theinvention for use in the treatment of a disease or condition susceptibleto treatment with an inhibitor of IL-1 (or IL_33) signalling.

Such conditions or disease states are well known in the art (seeDinarello et al., 2012, Nature Reviews 11:633-652 and Dinarello, 2014,Mol. Med. 20(suppl. 1):543-558)_(—) and include, but are not limited to,the following:

-   -   Rheumatoid arthritis, all types of juvenile arthritis including        systemic onset juvenile idiopathic arthritis (SOJIA),        osteoarthritis, familial cold auto-inflammatory syndrome (FCAS),        Muckle-Wells disease, neonatal onset multi-system inflammatory        disease (NOMID), familial Mediterranean fever (FMF), pyogenic        arthritis pyoderma gangrenosum and acne (PAPA) syndrome, adult        onset Still's disease, hyper IgD syndrome, type 2 diabetes        mellitus, macrophage activation syndrome, TNF        receptor-associated periodic syndrome, Blau disease, ankylosing        spondylitis, Sweets disease, lupus arthritis, Alzheimer's        disease, psoriasis, asthma, atherosclerosis, sarcoidosis, atopic        dermatitis, systemic lupus erythematosus, bullous pemphigoid,        type I diabetes mellitus, chronic obstructive pulmonary disease,        Helicobacter pylori gastritis, inflammatory bowel disease        (including ulcerative colitis and Crohn's disease), Hepatitis C,        ischaemia-reperfusion injury, multiple sclerosis, Neisserial or        pneumococcal meningitis, tuberculosis, Bechet's syndrome, septic        shock, graft versus host disease, asthma, type I diabetes,        Alzheimer's disease, atherosclerosis, adult T cell leukaemia,        multiple myeloma, periodontitis, obesity and obesity-related        diseases (for example, metabolic syndrome, cardiomegaly,        congestive heart failure, varicose veins, polycystic ovarian        syndrome, gastroesophageal reflux disease (GERD), fatty liver        disease, colorectal cancer, breast cancer, uterine cancer,        chronic renal failure, stroke and hyperuricemia), intervertebral        disc disease, irritable bowel syndrome, Schnitzler syndrome,        allergy/atopic dermatitis and gout.

Blockade of IL-1 signalling is also believed to beneficial in thetreatment of myocardial infarction. An extensive clinical trial iscurrently seeking to confirm the efficacy of IL1B antibody blockade(using Canakinumab) following myocardial infarction (the CANTOS trail;see Ridker et al., 2011, Am Heart Journal 162(4):597-605).

For such indications, it will be appreciated that a therapeutic benefitmay also be achieved using an antibody or antigen-binding fragment thatlacks ADCC activity, for example through inhibition of IL-1 (or IL-33)signalling associated with immune cells.

A thirteenth aspect of the invention provides the use of an antibody orantigen-binding fragment thereof according to the first aspect of theinvention in the preparation of a medicament for the treatment of adisease or condition susceptible to treatment with an inhibitor of IL-1(and/or IL-33 and/or IL-36) signalling.

A fourteenth aspect of the invention provides a method for the treatmentof a disease or condition susceptible to treatment with an inhibitor ofIL-1 (and/or IL-33 and/or IL-36) signalling in a subject, comprising thestep of administering to the subject an effective amount of an antibodyor antigen-binding fragment thereof according to the first aspect of theinvention.

A fifteenth aspect of the invention provides a method for theADCC-mediated treatment or augmentation of a disease or conditionsusceptible to treatment with an inhibitor of IL-1 (and/or IL-33 and/orIL-36) signalling in a subject, comprising the step of administering tothe subject an effective amount of an antibody or antigen-bindingfragment thereof according to the first aspect of the invention capableof inducing ADCC.

A sixteenth aspect of the invention provides an in vitro method for thedetection of cancer cells in a subject, the method comprising:

-   -   (a) providing a sample of cells (e.g. white blood        stem/progenitor cells or biopsy tissue) from a subject to be        tested;    -   (b) optionally, extracting and/or purifying the cells present in        the sample;    -   (c) contacting an antibody or antigen-binding fragment thereof        according to the first aspect of the invention with cells        present in the sample;    -   (d) determining whether the antibody polypeptide binds to the        cells        wherein the binding of the antibody polypeptide to the cells is        indicative of the presence of cancer cells in the tissue of a        subject.

A seventeenth aspect of the invention provides an in vitro method foridentifying a patient with cancer who would benefit from treatment withan antibody or antigen-binding fragment thereof according to the firstaspect of the invention, the method comprising:

-   -   (a) providing a sample of cancer cells (e.g. white blood        stem/progenitor cells or biopsy tissue) from a patient to be        tested;    -   (b) optionally, extracting and/or purifying the cells present in        the sample;    -   (c) contacting an antibody or antigen-binding fragment thereof        according to the first aspect of the invention with cells        present in the sample;    -   (d) determining whether the antibody polypeptide binds to the        cells        wherein the binding of the antibody polypeptide to the cancer        cells is indicative of a patient who would benefit from        treatment with an antibody or antigen-binding fragment thereof        according to the first aspect of the invention.

Persons skilled in the art will appreciate that there are many ways toperform such an assay. For example, the immunoassay could be eitherhomogeneous or, more preferably, heterogenous. The assay could also beperformed in either a competitive or, more preferably, a non-competitiveformat.

In one embodiment, IL1RAP expression on blood samples (leukemia) orbiopsies (solid tumours) from patients is measured using flow cytometryor immunohistochemistry, with expression above a threshold value beingindicative of a patient who would benefit from treatment with anantibody or antigen-binding fragment thereof according to the firstaspect of the invention.

In preferred embodiments of the above in vitro methods, step (d) isperformed by flow cytometry or ELISA. However, any assay suitable fordetecting antibody-antigen interactions in vitro may be used.

An eighteenth aspect of the invention provides a method for treating apatient with cancer, the method comprising administering to a subjectidentified as having cancer using a method according to the sixteenth orseventeenth aspects of the invention a therapeutic agent effective inthe treatment of said cancer. In one embodiment, the example therapeuticagent is an antibody polypeptide according to the first aspect of theinvention.

In one embodiment, the method comprises:

-   -   (a) arranging for a sample of cells (e.g. white blood        stem/progenitor cells or biopsy tissue) from a subject to be        tested for the presence of cancer cells expressing IL1RAP above        a threshold criteria using a method according to the sixteenth        or seventeenth aspect of the invention;    -   (b) selecting for treatment subjects whose sample of cells        tested in step (a) contains cancer cells with IL1RAP expression        above a threshold criteria; and    -   (c) administering to the subject selected in step (b) a        therapeutic agent effective in the treatment of said cancer, for        example an antibody polypeptide according to the first aspect of        the invention.

It will be appreciated by persons skilled in the art that cancer cellsexpressing IL1RAP may be identified using a probe capable of bindingspecifically to either:

-   -   (a) an IL1RAP polypeptide (e.g. an antibody or antigen-binding        fragment thereof according to the invention); or    -   (b) an IL1RAP polynucleotide transcript/mRNA (e.g. using an        oligonucleotide probe complementary in sequence to a region of        the IL1RAP polynucleotide transcript/mRNA).

Methods for performing such testing are well known in the art.

In a related embodiment, the method comprises:

-   -   (a) obtaining a sample of cells (e.g. white blood        stem/progenitor cells or biopsy tissue) from a subject    -   (b) testing said cells for the presence of cancer cells        expressing IL1RAP above a threshold criteria using a method        according to the sixteenth or seventeenth aspect of the        invention;    -   (c) selecting for treatment subjects whose sample of cells        tested in step (b) contains cancer cells with IL1RAP expression        above a threshold criteria; and    -   (d) administering to the subject selected in step (c) a        therapeutic agent effective in the treatment of said cancer, for        example an antibody polypeptide according to the first aspect of        the invention.

Optionally, steps (a) and (b) of the above embodiments may be repeatedfollowing a first period of treatment (for example, after one month) todetermine is the number of cancer cells with IL1RAP expression above athreshold criteria has reduced. If an inadequate reduction (e.g. noreduction) is observed, then the dose of therapeutic agent effective inthe treatment of said cancer is increased for a subsequent treatmentround. If no cancer cells with IL1RAP expression above a thresholdcriteria are detected, the treatment may be terminated.

A nineteenth aspect of the invention provides a in vitro diagnosticmethod for identifying a patient with cancer who would benefit fromtreatment with an antibody or antigen-binding fragment thereof of theinvention, said method comprising the steps of:

-   -   (a) contacting in vitro a sample of cells (e.g. white blood        stem/progenitor cells or biopsy tissue) from a subject to be        tested with a molecular probe capable of binding specifically to        an IL1RAP polypeptide, or to an IL1RAP polynucleotide        transcript, said probe being covalently bound to a moiety        capable of emitting photons; and    -   (b) detecting photons emitted from said moiety and forming an        image of the sample,        wherein localised emission of photons from said moiety is        indicative of said subject being a patient with cancer who would        benefit from treatment with an antibody or antigen-binding        fragment thereof of the invention.

Preferences and options for a given aspect, feature or parameter of theinvention should, unless the context indicates otherwise, be regarded ashaving been disclosed in combination with any and all preferences andoptions for all other aspects, features and parameters of the invention.For example, in one embodiment the invention provides an intact IgG1antibody comprising a heavy chain variable region having the amino acidsequence of SEQ ID NO:1 and a light chain variable region having theamino acid sequence of SEQ ID NO:2 for use in the treatment of AML.

The listing or discussion of an apparently prior-published document inthis specification should not necessarily be taken as an acknowledgementthat the document is part of the state of the art or is common generalknowledge.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

These, and other, embodiments of the invention will be betterappreciated and understood when considered in conjunction with the abovedescription and the accompanying drawings. It should be understood,however, that the above description, while indicating variousembodiments of the invention and numerous specific details thereof, isgiven by way of illustration and not of limitation. Many substitutions,modifications, additions and/or rearrangements may be made within thescope of the invention without departing from the spirit thereof, andthe invention includes all such substitutions, modifications, additionsand/or rearrangements.

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Binding to human IL-1RAP of the exemplary antibodies CAN01 andCAN03 in an indirect ELISA.

FIG. 2. Binding of exemplary antibodies of the invention (CAN01 andCAN03) to human CML cells. The graph shows the MFI value for KU812 cellsstained with IL1RAP-targeting monoclonal antibodies at a concentrationof 0.1 μg/mL.

FIG. 3. Ability of exemplary antibody CAN03 to block IL-1b signalling.

FIGS. 4A-4D. In vitro ADCC assay shows that exemplary antibody CAN01 andCAN03 induces specific cell killing of CML cells. (FIG. 4A) KU812,LAMA84, and BV173 cells were specifically killed by addition of 10 μg/mLCAN01. (FIG. 4B) The cell killing mediated by exemplary antibodies CAN01and CAN03 is dose dependent as shown on BV173 target cells. (FIG. 4C)Cell killing of primary cells from two CML blast crisis patients wasinduced by 1 μg/mL CAN01. (FIG. 4D) Cells from a third CML blast crisispatient carrying the T315I mutation were sensitive to the ADCC effectmediated by CAN01. Each experiment was performed at least twice with NKcells from different donors, and the presented data shows onerepresentative experiment from each.

FIG. 5. In vitro ADCC assay showing that the exemplary antibodies CAN01and CAN03 are efficient in inducing specific cell killing of melanomacells (SKMEL5 cell line). Already at 1 μg/mL, CAN01 and CAN03 show ahigh specific target cell killing. The experiment was performed at leasttwice with NK cells from different donors, and the presented data showsone representative experiment.

FIGS. 6A-6C. (FIG. 6A) Confocal images (one optical section, about 0.9μm thick) showing LAMA cells incubated for 2 hours with CAN01-AF488conjugated antibodies on ice, or at 37° C. for 2 hours, or beenincubated with CAN01-AF488 conjugated antibodies for 16 hours at 37 C.°.A clearly defined antibody binding to the cell membrane of the majorityof cells can be observed after 2 hours incubation on ice (“Ice 2 h)”.After incubation for 2 hours with CAN01-AF488 conjugated antibodies at37° C., in addition to membrane binding, antibodies have started toenter the cells (internalization) and are now localized also in thecytosol. After 16 hours of incubation at 37° C., the cell membranebinding is still present and the antibody internalization has producedaccumulation of CAN01-AF488 antibodies in the majority of cells. Scalebar (right image) represents 20 μm in all images. (FIG. 6B) Confocalimages (one optical section, about 0.9 μm thick) showing LAMA cellsincubated for 2 hours with CAN03-AF488 conjugated antibodies on ice, orat 37° C. for 2 hours, or been incubated with CAN03-AF488 conjugatedantibodies for 16 hours at 37 C.°. A clearly defined antibody binding tothe cell membrane of the majority of cells can be observed after 2 hoursincubation on ice (“Ice 2 h)”. After incubation for 2 hours withCAN03-AF488 conjugated antibodies at 37° C., in addition to membranebinding, antibodies have started to enter the cells (internalization)and are now localized also in the cytosol. After 16 hours of incubationat 37° C., the cell membrane binding is still present and the antibodyinternalization has produced accumulation of CAN03-AF488 antibodies inthe majority of cells. Scale bar (right image) represents 20 μm in allimages. (FIG. 6C) Control for the CAN03-specific binding andinternalization: The confocal images (one optical section, about 0.9 μmthick) show LAMA cells incubated with AF488 conjugated isotype controlantibody on ice or at 37° C. for 2 hours, or at 37 C.° for 16 hours. Theisotype control antibody showed no specific binding at any of theseconditions. Minor binding to cellular debris and necrotic cells wasnoted. Scale bar (right image) represents 20 μm in all images.

FIGS. 7A-7E. Treatment with CAN01 significantly reduces the leukemiaburden. (FIG. 7A) The frequency of leukemic cells in peripheral bloodwas lower in mice treated with exemplary antibody CAN01 compared toisotype control at day 36 after transplantation (0.10% vs. 0.69%,p<0.0043). (FIG. 7B) The platelet (PLT) count were lower with isotypethan with CAN01 (p=0.015). (FIG. 7C) At day 62 the mean frequency ofleukemic cells in peripheral blood was 46.5% in isotype treated mice butonly 3.8% with CAN01. (FIG. 7D) At time of sacrifice the frequency ofleukemic cells in the spleen was reduced with CAN01 (17.2% vs. 49.7%;p=0.0052). (FIG. 7E) The frequency of leukemic cells in bone marrow waslower in mice treated with CAN01 compared to isotype control (5.0% vs.59.5%; p=0.0001).

FIGS. 8A-8D. Treatment with CAN01 prolongs survival and reduces theleukemia burden. (FIG. 8A) The median survival for isotype treated micewas 37 days and 58 days for mice treated with CAN01 (p<0.0001) (FIG. 8B)At time of sacrifice the frequency of leukemic cells in the bone marrowwas reduced with CAN01 (29.2% vs. 55.9%; p=0.020). (FIG. 8C) The meanfrequency of leukemic cells in was lower in mice treated with CAN01compared to isotype control (31.7% vs. 43.1%). (FIG. 8D) Mice treatedwith CAN01 had significantly smaller spleens than mice given isotypecontrol (60 mg vs. 97 mg; p=0.032))

FIG. 9. Treatment with CAN01 significantly reduces the leukemia burdenin mice transplanted with primary human AML cells. The mean frequency ofleukemic cells in bone marrow was 0.001% in mice treated with CAN01 and0.126% in mice given control antibody.

FIGS. 10A-10C. Effects of CAN01, CAN03 and Isotype control on HEK-BlueIL-33/IL1β cells stimulated with (FIG. 10A) human IL-1α, (FIG. 10B)human IL-1β, and (FIG. 10C) human IL-33. Two experiments were performedwith duplicate samples, and represented as the individual values plusthe mean value as three individual data points.

FIGS. 11A-11C. Effects of CAN01, CAN03 and Isotype control on HEK-BlueIL-33/IL 1β cells stimulated with (FIG. 11A) murine IL-1α, (FIG. 11B)murine IL-1β, and (FIG. 11C) murine IL-33,

1β cells. Two experiments were performed with duplicate samples, andrepresented as the individual values plus the mean value as threeindividual data points.

EXAMPLES

A. Binding Affinity of Exemplary Antibodies of the Invention for IL1RAPProtein

(i) Biacore Study—Anti-IL1RAP Antibodies of Murine Origin

Materials & Methods

Goat anti-mouse IgG was immobilized on a CM5 chip according to thetechnical manual of capture kit and standard operation principle ofBIAcore T200 (Biacore Life Sciences, GE Healthcare Europe GmbH, Uppsala,Sweden).

The binding analysis cycle consisted of three steps: (i) capture of theligand on the chip surface by immobilized anti-mouse antibody; (ii)binding of the analyte to the captured ligand; and (iii) dissociation ofbound analyte.

The capture molecule surface was regenerated after each binding cycleusing the manufacturer's recommended conditions.

All binding cycles were run at 25° C.

After five cycles of start-up, each antibody (100 nM) was injected at aflow rate of 30 μl/min, for 120 s, at the start of the cycle; then theanalyte (100 nM) was injected at a flow rate of 30 μl/min, for 120 s,followed by monitoring the dissociation phase for 300 s.

Two exemplary antibodies of the invention (CAN01 and CAN03) were tested.

Results & Conclusions

Results are shown in Table 2 below:

TABLE 2 Measurement of K_(on), K_(off) and K_(D) Antibody ka (1/M · s)kd (1/s) KD (M) CAN01 2.34E+05 3.35E−04 1.43E−09 CAN03 2.26E+05 7.25E−053.21E−10

(ii) ELISA Study—Anti-IL1RAP Antibodies of Murine Origin

Materials & Methods

An indirect ELISA assay was performed. All samples were analysed induplicate. Nunc-MaxiSorp 96 Micro Well™ Plates were coated with 100 ngof recombinant hIL1RAP 21-367 (100 μl/well) diluted in 0.01M PBS, pH7.4, and incubated overnight at 4° C. Plates were washed with ELISAwashing buffer (0.01M PBS, 0.05% Tween 20, pH 7.4) followed by ablocking step using 150 μl/well of ELISA blocking solution (PBS, 0.5%BSA, 0.05% Tween 20, pH 7.4). After 1 h incubation at room temperature(RT) on agitation the plates were washed again using ELISA washingbuffer. Samples were diluted in three fold serial dilution (ranging from1000 ng/ml to 0.5 ng/ml) in ELISA blocking solution and then transferredto the ELISA plate, 100 μl/well. Plates were incubated at RT for 1 h onagitation and then washed with ELISA washing solution. 100 μl/well ofrabbit anti-mouse IgG conjugated to Alkaline Phosphatase (DAKO, 1:1000)was added and incubated 1 hour at RT on agitation. The plates werewashed followed by addition of substrate (4-Nitrophenyl phosphatisedisodium salt hexahydrate, SIGMA, 1 mg/ml), 100 μl/well. The plates werethereafter incubated at RT on agitation and absorbance at 405 nmmeasured consecutively for 30 min. Absorbance at 0 min was taken asbackground signal.

Results & Conclusions

Results are shown in FIG. 1

The exemplary antibodies of the invention, CAN01 and CAN03, were bothfound to possess a high binding signal for human IL1RAP.

B. Flow Cytometry Study of the Binding of Exemplary Antibodies of theInvention to IL1RAP-Expressing Cells

Materials & Methods

Chronic myeloid leukemia (CML) cell line KU812 cells were stained withantibodies raised against IL1RAP or a relevant isotype control. Fordetection, a secondary anti-mIg-APC was used.

Two exemplary antibodies of the invention (CAN01 and CAN03) were testedalong with five comparator anti-IL1RAP antibodies (CAN02, CAN05, CAN07,CAN08 and CAN09). An isotype negative control antibody was alsoincluded.

Results & Conclusions

Staining of IL1RAP-expressing KU812 leukemia cells reveals a higher meanfluorescence intensity (MFI) for CAN01 and CAN03 compared to the isotypecontrol and other comparator antibodies targeting IL1RAP (FIG. 2).

C. Epitope/Domain Mapping of Exemplary Antibodies of the Invention

Materials & Methods

In order to understand where the different antibody clones bind on theIL1RAP, a structural analysis of the protein was performed revealingthat the extracellular part of the receptor could be divided into threedistinct domains hereafter referred to as domains 1, 2 and 3 (D1, D2,D3) (see Wang et al., 2010, Nature Immunology, 11:905-912, thedisclosures of which are incorporated herein by reference). In order todetermine the domain-binding pattern for the different antibody clones,a series of receptor constructs were generated and binding to thesetested in an ELISA assay.

An indirect ELISA assay was performed. All samples were analysed induplicate. Nunc-MaxiSorp 96 Micro Well™ Plates were coated with 100 ngof Rec hIL1RAP Domain123 (aa21-367) (positive control), Rec hIL1RAPDomain12 (aa21-234), Domain1 (aa21-134) or Rec hIL1RAP Domain3(aa235-367) (100 μl/well) diluted in 0.01M PBS, pH 7.4, and incubatedovernight at 4° C. Plates were washed with ELISA washing buffer (0.01MPBS, 0.05% Tween 20, pH 7.4) followed by a blocking step using 150μl/well of ELISA blocking solution (PBS, 0.5% BSA, 0.05% Tween 20, pH7.4). After 1 h incubation at room temperature (RT) on agitation theplates were washed again using ELISA washing buffer. CAN01, CAN03,CAN05, CAN07, CAN08 and KMT-1 (positive control) were diluted in threefold serial dilution (ranging from 1000 ng/ml to 0.5 ng/ml) in ELISAblocking solution and then transferred to the ELISA plate, 100 μl/well.Plates were incubated at RT for 1 h on agitation and then washed withELISA washing solution. 100 μl/well of rabbit anti-mouse IgG conjugatedto Alkaline Phosphatase (DAKO, 1:1000) was added and incubated 1 hour atRT on agitation. The plates were washed followed by addition ofsubstrate (4-Nitrophenyl phosphatise disodium salt hexahydrate, SIGMA, 1mg/ml), 100 μl/well. The plates were thereafter incubated at RT onagitation and absorbance at 405 nm measured consecutively for 30 min.Absorbance at 0 min was taken as background signal.

Two antibodies of the invention (CAN01 and CAN03) were tested along withfive comparator anti-IL1RAP monoclonal antibodies (CAN02, CAN05, CAN07,CAN08, together with a polyclonal anti-IL1RAP antibody (KMT-1) as apositive control).

Results & Conclusions

The exemplary antibodies of the invention, CAN01 and CAN03, were foundto bind within domain 3 of IL1RAP.

The domain mapping data can be found summarized in the Table 3 below.

TABLE 3 Epitope mapping of exemplary anti-IL1RAP antibody clones.Domain123 Domain12 Domain1 Domain3 Suggested Clone (aa21-367) (aa21-234)(aa21-134) (aa235-367) epitope CAN03 + + D3 CAN05 + + + D1 CAN07 + + D3CAN08 + + D3 CAN01 + + D3 CAN02 + nd* KMT-1 + + + + polyclonal nd* = notdetermined as epitope mapping data could not clearly identify specificdomain for these constructs, which may be attributed to binding to astructural epitope containing sequence elements from more than onedomain, e.g. D2-D3 junction.

D. Specificity/Cross-Reactivity of Exemplary Antibodies of the Invention

Materials & Methods

An important feature of a good lead candidate antibody is that itcross-reacts with equal or near-equal potency to the homologous proteinin a relevant toxicology species. According to the general regulatoryguidelines, binding to one rodent and one non-rodent would be thepreferred scenario, but for antibodies this is rarely the case, andinstead many labs struggle to identify any relevant toxicology speciesexcept for primates.

For the present study, cross reactivity to non-human primates likeMacaca mulatta (rhesus) or Macaca fascicularis (cynomolgus) was expectedsince the IL1RAP protein in these species share 99% homology to thehuman IL1RAP protein.

A number of potential lead antibodies were selected and tested forbinding to recombinant M. fascicularis IL1RAP (aa21-367) in an ELISAassay.

Two antibodies of the invention (CAN01 and CAN03) were tested along withsix comparator anti-IL1RAP monoclonal antibodies (CAN02, CAN07, CAN08,CAN09, Mab676 from R&D, and a polyclonal anti-IL1RAP antibody (KMT-1).

Results & Conclusions

Surprisingly, several of the comparator anti-IL1RAP antibodies testedwere found not to cross-react with cynomolgus IL1RAP, amongst them thecommercial reference antibody mAb676 from R&D, Table 4.

TABLE 4 Binding to cynomolgus IL1RAP (Values in bold denotes clonesidentified to cross-react with IL1RAP from M. fascicularis) Binding torec. M. fascicularis Clone IL1RAP (OD₄₀₅) CAN01 0.324 CAN02 0.014 CAN090.022 CAN03 0.870 CAN07 0.111 CAN08 0.375 mAb676 (R&D) 0.037 KMT-1 0.481

E. Inhibition of IL-1 Signalling by Exemplary Antibody of the Invention

(i) Effect in HEK-Blue Cell Line

Materials & Methods

As IL1RAP is a functional part of the IL-1 receptor complex, antibodiesbinding to IL1RAP may also inhibit IL-1 signalling. Since a number oftumour cell types have been shown to use IL-1 as a growth factor, thismay be an important additional mechanism for mediating anti-tumoureffects.

In order to test for the capability of potential lead candidateantibodies to block IL-1 signalling, an IL-1 dependent reporter geneassay was set up. HEK-Blue cells (InvivoGen) respond to IL-1 signallingby the release of alkaline phosphatase that can be quantified by acolorometric assay. To test the inhibitory capacity of the leadcandidates HEK-Blue cells were plated at 50 000 cells/well and incubatedwith the test antibodies 30 minutes prior to stimulation with IL-1β. Thecells were then incubated at 37° C. o/n before measuring the amount ofalkaline phosphatase released. Antibodies were also tested for potentialagonistic effects by incubating the cells in the presence of a highconcentration of antibody (10 mg/ml) in the absence of additionalstimuli. Any IL-1R agonistic effects would thus be recorded as a releaseof alkaline phosphatase.

One antibody of the invention (CAN03) was tested along with an isotypenegative control antibody.

Results & Conclusions

As depicted in FIG. 3, the exemplary antibody CAN03 induced a pronouncedinhibition of IL-1 signalling. The tested candidate showed no agonisticeffect.

F. ADCC Effect of Exemplary Antibodies of the Invention in ChronicMyeloid Leukemia (CML) Cell Lines

Materials & Methods

Chronic myeloid leukemia (CML) cell lines KU812, LAMA84 and BV173, orprimary cells from three patients with CML in blast crisis were used astarget cells in the in vitro antibody dependent cellular cytotoxicity(ADCC) assay. Briefly, target cells were labelled with PKH26(Sigma-Aldrich, St Louis, Mo.) according to manufacturer's instructions,and seeded into a 96-well plate at a density of 5,000-10,000 cells perwell. Subsequently, the exemplar antibody of the invention, CAN01, orisotype control antibody was added to wells in different concentrationsand incubated for 30 min before 100,000 NK effector cells were added toeach well. NK-cells were extracted from healthy volunteers afterinformed consent by using an NK-cell negative cell isolation kitaccording to manufacturer's instructions (Miltenyi Biotech, BergischGladbach, Germany). A non-specific human IgG1 antibody was used as anisotype negative control in the experiments (Eureka Therapeutics,Emeryville, Calif.). The degree of cell death was assessed by detectionof 7-AAD positive cells using a FACS CANTO flow cytometer (BD). Eachexperiment was performed at least twice with NK cells from differentdonors.

Results & Conclusions

The in vitro ADCC assay shows that the exemplary antibody of theinvention, CAN01, directs NK-cells to kill CML cell lines KU812, LAMA84and BV173 to a higher degree than the isotype control (FIG. 4A). A dosetitration of CAN01 and CAN03 using BV173 target cells shows that theeffect on cell killing is dose dependent with a higher degree of cellkilling with increasing concentrations (FIG. 4B). Chronic myeloidleukemia that has progressed into blast crisis display only transienteffect to treatment with tyrosine kinase inhibitors and thus imposes amajor treatment problem. The ADCC assay with primary cells from twoindividual CML blast crisis patients shows that these cells weresensitive to the cellular cytotoxicity induced by CAN01 and NK-cells(FIG. 4C). In addition, primary cells from a third CML blast crisispatient harbouring the T315I mutation that cause resistance to severaltyrosine kinase inhibitors display similar sensitivity (FIG. 4D).Altogether, the experiments show that CAN01 has the ability to directNK-cells to specific cell killing of CML cell lines as well as primaryblast crisis CML cells, and that the cytotoxic effect induced by CAN01is dose dependent.

G. ADCC Effect of Exemplary Antibodies of the Invention in Melanoma CellLines

Materials & Methods

The malign melanoma cell line SKMEL-5 was used as a target for in vitroantibody dependent cellular cytotoxicity (ADCC) assay. Briefly, targetcells were labelled with PKH26 (Sigma-Aldrich, St Louis, Mo.) accordingto manufacturer's instructions, and seeded into a 96-well plate at adensity of 5,000-10,000 cells per well. Subsequently, the test antibodyor isotype control antibody were added to wells in differentconcentrations and incubated for 30 min before 100,000 NK effector cellswere added to each well. NK-cells were extracted from healthy volunteersafter informed consent by using an NK-cell negative cell isolation kitaccording to manufacturer's instructions (Miltenyi Biotech, BergischGladbach, Germany). A non-specific human IgG1 antibody was used ascontrol in the experiments (Eureka Therapeutics, Emeryville, Calif.).The degree of cell death was assessed by detection of 7-AAD positivewithin PKH26 positive cells using a FACS CANTO flow cytometer (BD). Eachexperiment was performed at least twice with NK cells from differentdonors.

Results & Conclusions

The in vitro ADCC assay showed that CAN01 and CAN03 direct NK-cells tokilling of the SKMEL-5 cell line to a much higher degree than a matchingisotype control (FIG. 5).

H. Internalisation of Exemplary Antibodies of the Invention

Materials & Methods

Cells and Culture Conditions: LAMA-84 cells, a cell-line establishedfrom a patient with chronic myeloid leukemia in blast crisis, wereobtained from DSMZ (Braunschweig, Germany) and cultured according to therecommendation by the supplier. Briefly, cells were cultured in RPMI1640 with 10% FBS, 1% Glutamine and 1% Penicillin/Streptomycin in 5%CO₂, 37° C. Cell cultures were split to a density of 0.5×10⁶ cells/mlevery 2-3 days. Cells were used for up to 12 passages after they werereceived from DSMZ.

Cells from the suspension cell-line LAMA-84 were washed once inphosphate buffered saline (PBS) supplemented with 1% Bovine serumalbumin (BSA) and resuspended in PBS-BSA supplemented with 5% human AB+serum from Sigma and incubated for 5 minutes at room temperature (RT).The AlexaFluor488 (AF488) labelled IL-1RAP selective antibodies CAN01,CAN03 or isotype matched control antibody, was added to a finalconcentration of 10 μg/ml. Cells were placed (incubated) on ice or at37° C. for 2 or 16 hours.

For the image analysis with confocal microscopy (LSM 510 Meta Zeissconfocal microscope), cells were washed twice in PBS-1% BSA, werebriefly spun down followed by resuspension in 3% paraformaldehyde (inPBS) fixation for 20 minutes (at 4 C.°). Cells were then spun down,resuspended in PBS containing 0.001% Triton X-100 (PBS-TX) and a nuclearmarker (DAPI), and were let to incubate for 5 minutes at RT. After abrief centrifugation cells were resuspended in PBS-TX and placed inglass-bottomed microscope wells. Cells were then let to adhere for onehour. Image data were collected via confocal scanning of cells providinghigh-resolution images of AlexaFluor488 fluorescence in thin opticalsections through the centre of cells (depicted by nuclear marker).Further analyses of antibody binding to cell membrane and/orinternalized antibodies were performed via software image analyses(Zeiss Zen2010).

Results & Conclusions

The structural relation of CAN01 and CAN03 binding to the cell membraneand its capacity to enter the cells (“internalize”) was demonstratedwith high resolution imaging data recorded by means of confocal laserscanning microscopy, and by image analyses of this data.

Image data representations are shown in FIG. 6.

I. Therapeutic Efficacy In Vivo of an Exemplary Antibody of theInvention

Materials & Methods

Unconditioned NOD/SCID mice were engrafted with lethal doses of MA9Rascells, previously generated by transformation of human umbilical cordblood CD34⁺ cells by retroviral integration of cDNAs directing theexpression of an MLL/AF9 fusion and an activated NRAS gene. Leukemicmice were treated with the exemplary CAN01 antibody targeting IL1RAP, ora corresponding isotype control antibody. The antibodies wereadministered by intraperitoneal injections twice weekly throughout theexperiment with first treatment given day three after transplantation.Each dose of antibody was 500 μg, except for the first that was given asa bolus of 1000 μg. Mice were sacrificed upon signs of severe disease asjudged by hunchback, untidy fur, and decreased mobility, or due to solidtumours.

Results & Conclusions

Immunodeficient mice were engrafted with human leukemic cells andtreated with CAN01, a monoclonal antibody targeting IL1RAP. Thefrequency of leukemic cells in peripheral blood was significantlyreduced at day 35 after transplantation, and the platelet countsremained normal in mice given CAN01 compared to isotype control antibodyindicating a more functional haematopoiesis (FIG. 7A-B). At 62 daysafter transplantation the isotype treated mice had a high frequency ofleukemic cells in peripheral blood (FIG. 7C). CAN01 treatment resultedin a significant reduction of leukemic cells in spleen and bone marrow(FIG. 7D-E). We conclude that anti-IL1RAP immunotherapy reduces humanleukemia in peripheral blood, bone marrow, and spleen, in the MA9Rasxenograft model. The results support anti-IL1RAP immunotherapy as a newpromising therapeutic strategy for AML.

J. Therapeutic Efficacy In Vivo of an Exemplary Antibody of theInvention

Materials & Methods

Unconditioned NOD/SCID mice were engrafted with BV173 CML cells, whichharbour the BCR/ABL fusion gene. Leukemic mice were treated with theexemplary CAN01 antibody targeting IL1RAP, or a corresponding isotypecontrol antibody. The antibodies were administered by intraperitonealinjections twice weekly for a maximum of 13 doses, with first treatmentgiven day two and the last one day 45 after transplantation. Each doseof antibody was 500 μg, except for the first that was given as a bolusof 1000 μg. Mice were sacrificed upon signs of severe disease as judgedby hunchback, weight loss, and decreased mobility.

Results & Conclusions

Immunodeficient mice were engrafted with human CML cells and treatedwith CAN01, a monoclonal antibody targeting IL1RAP. Mice given isotypecontrol had a median survival of 37 days (range 33-38 days) whereas micetreated with CAN01 survived significantly longer (median 51 days),despite that the last treatment was administered at day 45 (FIG. 8A).Two CAN01 treated mice were still alive and apparently healthy atsacrifice day 101 after transplantation. CAN01 treatment resulted in asignificant reduction of leukemic cells in the bone marrow (FIG. 8B). Inthe spleen there was a trend towards reduced leukemic frequency withCAN01 (FIG. 8C). Mice treated with CAN01 had significantly smallerspleens than isotype treated mice (FIG. 9D). We conclude thatanti-IL1RAP immunotherapy prolongs survival and reduces human leukemiain the BV173 xenograft model. The results support anti-IL1RAPimmunotherapy as a new promising therapeutic strategy for CML.

K. Therapeutic Efficacy In Vivo of an Exemplary Antibody of theInvention

Materials & Methods

Mononuclear cells were isolated from a bone marrow aspirate taken froman AML patient at diagnosis. The cells were transplanted by intravenousinjection into unconditioned NOD/SCID mice. Starting three days aftertransplantation, mice were treated with anti-IL1RAP CAN01 or acorresponding mIgG2a isotype control antibody. The antibodies wereadministered by intraperitoneal injections twice weekly throughout theexperiment. Each dose of antibody was 100 μg, except for the first thatwas given as a bolus of 500 μg. Mice were sacrificed 28 days aftertransplantation, and the bone marrow was analysed for frequency of humanleukemic cells as detected by expression of CD45 and CD33 on flowcytometry.

Results & Conclusions

Immunodeficient mice were engrafted with primary human AML cells andtreated with CAN01, a monoclonal antibody targeting IL1RAP. At sacrifice28 days after transplantation, the frequency of leukemic cells in bonemarrow was significantly reduced with CAN01 compared to isotype control(0.001% vs. 0.126%, p<0.0001; FIG. 9). We conclude that anti-IL1RAPimmunotherapy reduces leukemia in bone marrow in mice engrafted withprimary human AML cells. The results add strength to anti-IL1RAPimmunotherapy as a new promising therapeutic strategy for AML.

L. In Vitro ILIRAP Dependent Blocking of Human IL-1α-, IL-1β- andIL-33-Mediated Signalling in HEK-Blue IL-33/IL-1β Cells. Potency ofAntibodies CAN01 and CAN03.

The following experiments were performed to determine whether twoexemplary antibodies of the invention (CAN01 and CAN03) were able toinhibit IL-1alpha and beta dependent signalling in the reporter geneassay using HEK-Blue IL-33/IL-1β cells.

Materials

Preparation of Antibodies

Exemplary antibodies of the invention (CAN01 and CAN03) together with anisotype control (mIgG2a) were provided by Innovagen AB (Lund, Sweden).

All antibodies were diluted in PBS for testing. Working solutions forreporter gene assay, HEK-Blue IL-33/IL-1β were made in PBS at 20 timesat the final assay concentration. The final assay volume was 200 μl(containing 10 μl antibody or diluent (PBS)+180 μl cells+10 μl ligand)Final assay concentrations of antibodies were as follows; 100 to 0.01 nM(by serial dilutions in 3-fold dilutions steps).

Preparation of Human Ligands (IL-1α, IL-1β and IL-33)

Human ligands, IL-1α. IL-1β and IL-33 were diluted in PBS to a stockconcentration of 10 μg/mL. Stock concentrations of 10 μg/mL of theligands were stored in aliquots at −80° C. until use. For the reportergene assay, the ligands were used at a final concentration of 0.3 ng/mL,the concentration that gave 70-80% of the maximal effect in the assay asdetermined in a pre-test experiment.

Dilution of Ligands to Final Assay Concentration

-   -   Stock of ligands 10 μg/mL was diluted 1:100 (2 μl+198 μl        Selection medium, see below)=100 ng/mL.    -   100 ng/mL was diluted 1:16.67 (150 μl+2350.5 μl Selection        medium, see below)=6 ng/mL.    -   6 ng/mL was diluted 1:20 in the assay; 10 μl of LPS 6 ng/mL+190        μl cells and compound/well to yield a final concentration of 0.3        ng/mL.

Cell Line

HEK-Blue IL-33/IL-1β cells, generated by stable transfection ofHEK-Blue™ IL-1β cells with the IL1RL1, were used as IL-33/IL-1β sensorcells (Cat no. hkb-IL-33, InvivoGen, San Diego, US).

Methods

Study Design

Stock solutions of CAN01, CAN03, and Isotype control were prepared inPBS. Working solutions for reporter gene assay, HEK-Blue IL-33/IL-1βwere made in PBS at 20 times at the final assay concentration. The finalassay volume was 200 μl (containing 10 μl antibody or diluent (PBS)+180μl cells+10 μl ligand). Final assay concentrations of antibodies were asfollows; 100 to 0.01 nM (by serial dilutions in 3-fold dilutions steps.In control wells (stimulated/unstimulated cells) antibodies werereplaced by 10 μl PBS.

Culturing and Stimulation of HEK-Blue IL-33/IL-1β Cells

As IL1RAP is a functional part of the IL-1 receptor complex, antibodiesbinding to IL1RAP have the potential to inhibit IL-1 signalling. Sincetumour cells have been reported to use IL1RAP dependent ligands such asIL-1α, IL-1β and IL-33 as a growth factor, blocking this signal mayprovide an important mechanism for mediating anti-tumour effects (eitherseparately or combined with an ADCC effect). In order to test for thecapability of antibodies to block IL-1 signalling, an IL-1 dependentreporter gene assay was set up. HEK-Blue IL-33/IL-1β cells (InvivoGen)respond to IL-1 signalling by the release of alkaline phosphatase thatcan be quantified by a colorimetric assay. To test the inhibitorycapacity of the lead candidates HEK-Blue cells were plated at 50 000cells/well and incubated with the test antibodies 45 minutes prior tostimulation with IL-1α, IL-1β, and IL-33 in a final concentration of 0.3ng/ml for each ligand. Final assay concentrations of antibodies were 100nM-0.01 nM. In control wells, antibodies were replaced by PBS. The cellswere incubated at 37° C. o/n before measuring the amount of alkalinephosphatase released by QUANTI-Blue-Medium for detection andquantification of alkaline phosphatase.

The HEK-Blue IL-33/IL-1β cells were thawed and cultured in DMEM, 10% FCS(HI) and PEST and Normocin for two passages. After two passages thecells were cultured with selection antibiotics (Zeocin, HygroGold andBlasticidin) added to the medium above for at least one passage beforethe experiments, as well as during the experiments. HygroGold isrequired to maintain the IL-1β specificity to the cell line andBlasticidin and Zeocin are required to maintain the plasmids encodingIL1RL1 and SEAP respectively. The experiments were run on cells of 70%confluency. The cells were split 2-3 times/week, or when they hadreached 80-90% confluence.

The ligands were titrated in a dose range from 30 ng/ml to 0.001 ng/ml.To generate a good assay for testing the antibodies ability to affectthe IL-1 signalling (stimulate or inhibit the amount of alkalinephosphatase release) a concentration that resulted in a robust signal onthe linear slope of the dose-response curve is preferred. For thissystem a concentration of 0.3 ng/ml of each ligand was selected. Twoindividual experiments were performed with individual dilution series ofthe antibodies. Ligands were also diluted separately for each experimentand cells from different passages were used.

Evaluation of Results

Raw data were converted to % inhibition using equation 1:

% inhibition=(1−(A−B)/(C−B))×100

-   -   wherein:    -   A=Ligand activity with compound dissolved in PBS added    -   B=Negative control, No Ligand, only PBS (vehicle)    -   C=Positive control, Ligand with PBS (vehicle),

IC50 represents the concentration yielding 50% inhibition of the maximalresponse.

EC50 represents the concentration yielding an inhibition representing50% inhibition with respect to calculated values for the top and bottomof the curve.

Results & Conclusions

Experiments were set up elucidate the effect of two test antibodies(CAN03 and CAN01) in a reporter gene test system of HEK-Blue IL-33/IL-βcells stimulated with three different ligands (hIL-1α, hIL-1β andhIL-33) to release alkaline phosphatase. As a comparator and reference,an Isotype control was used. Cells prepared from two individual passageswere used. All experiments were performed using duplicate cultures.

In a first experiment, the effect of the three different ligands onHEK-Blue IL-33/IL-β cells were evaluated to create a relevant testsystem. The ligands were titrated from 30 ng/ml to 0.001 ng/ml and 0.3ng/ml was selected as an appropriate concentration for all ligands inthe test system. HEK-Blue IL-33/IL-1β cells were prepared from twodifferent passages. Cells were stimulated in the presence of 0.3 ng/mlligands for 16 hrs, and the supernatant harvested and assessed formeasuring the amount of alkaline phosphatase released.

As demonstrated in FIG. 10, CAN03 demonstrated an inhibitory effect toIL1RAP signalling by inhibiting the release of alkaline phosphatase withall ligands in the two experiments. CAN03 is able to fully inhibitIL-1α-mediated and IL-1β-mediated signalling. IL-33 induced signallingis blocked by CAN03 is to at least 95%. In contrast, CAN01 and theisotype control did not show any inhibitory effect.

Table 5 below summarises the potency for all tested antibody candidates.

TABLE 5 IC50/EC50 (nM) mean values for the effect of CAN03, CAN01 andIsotype control on HEK-Blue IL-33/IL-1β cells and three differentligands (n = 2) Ligand IL-1α Ligand IL-1β Ligand IL-33 AntibodyIC50/EC50 (nM) IC50/EC50 (nM) IC50/EC50 (nM) CAN03 37.34/37.6010.08/10.46 30.40/30.51 CAN01 NA/NA NA/NA NA/NA Isotype NA/NA NA/NANA/NA control

M. In Vitro IL1RAP Dependent Blocking of Murine IL-1α-, IL-1β- andIL-33-Mediated Signalling in HEK-Blue IL-33/IL-1β Cells. Potency ofAntibodies CAN01 and CAN03.

The following experiments were performed (i) to confirm that the murineligands can stimulate signalling through human receptor complexes, and(ii) to determine whether two exemplary antibodies (CAN01 and CAN03)were able to inhibit such signals.

Materials

Preparation of Antibodies

Exemplary antibodies of the invention (CAN01 and CAN03) together withtwo isotype controls (mIgG2a and hIgG1/kappa) were provided by InnovagenAB (Lund, Sweden).

All antibodies were diluted in PBS for testing. Working solutions forreporter gene assay, HEK-Blue IL-33/IL-1β were made in PBS at 20 timesat the final assay concentration. The final assay volume was 200 μl(containing 10 μl antibody or diluent (PBS)+180 μl cells+10 μl ligand)Final assay concentrations of antibodies were as follows; 100 to 0.01 nM(by serial dilutions in 3-fold dilutions steps).

Preparation of Human Ligands (IL-1α, IL-1β and IL-33)

Murine ligands, IL-1α. IL-1β and IL-33 were diluted in PBS to a stockconcentration of 10 μg/mL. Stock concentrations of 10 μg/mL of theligands were stored in aliquots at −80° C. until use. For the reportergene assay, the ligands were used at a final concentration of 10 ng/mLfor mIL-1α, 100 ng/mL for mIL-1β and 2 ng/mL for mIL-33; theseconcentrations gave 70-80% of the maximal effect in the assay asdetermined in a pre-test experiment.

Dilution of Ligands to Final Assay Concentration

(a) mIL-1α, 10 ng/mL

-   -   Stock of ligands 10 μg/mL was diluted 1:50 (20 μl+980 μl        Selective medium, see below)=200 ng/mL    -   200 ng/mL was diluted 1:20 in the assay; 10 μl of LPS 200        ng/mL+190 μl cells and compound/well to yield a final        concentration of 10 ng/mL

(b) mIL-1β, 100 ng/mL

-   -   Stock of ligands 10 μg/mL was diluted 1:5 (200 μl+800 μl        Selective medium, see below)=2000 ng/mL    -   2000 ng/mL was diluted 1:20 in the assay; 10 μl of LPS 2000        ng/mL+190 μl cells and compound/well to yield a final        concentration of 100 ng/mL

(c) mIL-33, 2 ng/mL

-   -   Stock of ligands 10 μg/mL was diluted 1:250 (10 μl+2490 μl        Selective medium, see below)=40 ng/mL    -   40 ng/mL was diluted 1:20 in the assay; 10 μl of LPS 200        ng/mL+190 μl cells and compound/well to yield a final        concentration of 2 ng/mL

Cell Line

HEK-Blue IL-33/IL-1β cells, generated by stable transfection ofHEK-Blue™ IL-1β cells with the IL1RL1, were used as IL-33/IL-1β sensorcells (Cat no. hkb-IL-33, InvivoGen, San Diego, US).

Methods

Study Design

Stock solutions of CAN01, CAN03 and the two isotype controls wereprepared in PBS. Working solutions for reporter gene assay, HEK-BlueIL-33/IL-1β were made in PBS at 20 times at the final assayconcentration. The final assay volume was 200 μl (containing 10 μlantibody or diluent (PBS)+180 μl cells+10 μl ligand). Final assayconcentrations of antibodies were as follows; 100 to 0.01 nM (by serialdilutions in 3-fold dilutions steps. In control wells(stimulated/unstimulated cells) antibodies were replaced by 10 μl PBS.

Culturing and Stimulation of HEK-Blue IL-33/IL-1β Cells

As IL1RAP is a functional part of the IL-1 receptor complex, antibodiesbinding to IL1RAP have the potential to inhibit IL-1 signalling. Sincetumour cells have been reported to use IL1RAP dependent ligands such asIL-1α, IL-1β and IL-33 as a growth factor, blocking this signal mayprovide an important mechanism for mediating anti-tumour effects (eitherseparately or combined with an ADCC effect). In order to test for thecapability of antibodies to block IL-1 signalling, an IL-1 dependentreporter gene assay was set up. HEK-Blue IL-33/IL-1β cells (InvivoGen)respond to IL-1 signalling by the release of alkaline phosphatase thatcan be quantified by a colorimetric assay. To test the inhibitorycapacity of the lead candidates HEK-Blue cells were plated at 50 000cells/well and incubated with the test antibodies 45 minutes prior tostimulation with murine IL-1α, IL-1β, and IL-33 in a final concentrationto give optimal stimulation. Final assay concentrations of antibodieswere 100 nM-0.01 nM. In control wells, antibodies were replaced by PBS.The cells were incubated at 37° C. o/n before measuring the amount ofalkaline phosphatase released by QUANTI-Blue-Medium for detection andquantification of alkaline phosphatase.

The HEK-Blue IL-33/IL-1β cells were thawed and cultured in DMEM, 10% FCS(HI) and PEST and Normocin for two passages. After two passages thecells were cultured with selection antibiotics (Zeocin, HygroGold andBlasticidin) added to the medium above for at least one passage beforethe experiments, as well as during the experiments. HygroGold isrequired to maintain the IL-1β specificity to the cell line andBlasticidin and Zeocin are required to maintain the plasmids encodingIL1RL1 and SEAP respectively. The experiments were run on cells of 70%confluency. The cells were split 2-3 times/week, or when they hadreached 80-90% confluence.

The ligands were titrated in a dose range from 300 ng/ml to 0.01 ng/ml.To generate a good assay for testing the antibodies ability to affectthe IL-1 signalling (stimulate or inhibit the amount of alkalinephosphatase release) a concentration that resulted in a robust signal onthe linear slope of the dose-response curve is preferred. For thissystem a concentration of 10 ng/mL for mIL-1α, 100 ng/mL for mIL-1β and2 ng/mL for mIL-33 were selected.

Evaluation of Results

Raw data were converted to % inhibition using equation 1:

% inhibition=(1−(A−B)/(C−B))×100

-   -   wherein:    -   A=Ligand activity with compound dissolved in PBS added    -   B=Negative control, No Ligand, only PBS (vehicle)    -   C=Positive control, Ligand with PBS (vehicle),

IC50 represents the concentration yielding 50% inhibition of the maximalresponse.

EC50 represents the concentration yielding an inhibition representing50% inhibition with respect to calculated values for the top and bottomof the curve.

Results & Conclusions

Experiments were set up elucidate the cross-reactivity of IL1RAPsignalling of murine ligands towards the human receptors.

In a first set of experiments, the three murine ligands IL-1 alpha, IL-1beta and IL-33 were tested for their ability to stimulate HEK BlueIL-33/IL-1β. All three murine ligands were able to stimulate the releaseof alkaline phosphatase, although with a significantly reduced effect ascompared to their human equivalents.

In this first experiment, the effect of the three different ligands onHEK-Blue IL-33/IL-β cells was also evaluated, to create a relevant testsystem. The ligands were titrated from 100 ng/ml to 0.001 ng/ml and 10ng/mL for mIL-1α, 100 ng/mL for mIL-1β and 2 ng/mL for mIL-33 wereselected. The drop in efficacy was greatest for IL-1β (around 300-fold),and smallest for IL-33 (around 8 fold).

The effect of the test antibodies to inhibit the signal induced by themurine ligands was subsequently tested (see FIG. 11). As a comparisonand reference, two isotype control antibodies were used (Human IgG1 andmurine IgG2a). HEK-Blue IL-33/IL-1β cells were prepared. Cells werestimulated in the presence of ligands for 16 hrs, and the supernatantharvested and assessed for measuring the amount of alkaline phosphatasereleased. Cells prepared from two individual passages were used. Allexperiments were conducted using duplicate cultures.

CAN03 inhibited IL1RAP-dependent signalling mediated by all three murineligands, as determined by release of alkaline phosphatase. CAN01 hadonly a limited inhibitory effect on IL1RAP-dependent signalling. None ofthe isotype controls showed any inhibitory effect. Due to the lack ofplateau at the top of the curve for ligands IL-1β and IL-33, no IC50 andEC50 values could be calculated for CAN01.

Table 6 below summarises the potency for all tested antibody candidates.

TABLE 6 IC50/EC50 (nM) mean values for the effect of CAN01, CAN03, MouseIgG2a (Isotype control) and Human IgG1/kappa (Isotype control) (n = 1)Ligand IL-1 alpha Ligand IL-1 beta Ligand IL-33 Antibody IC50/EC50 (nM)IC50/EC50 (nM) IC50/EC50 (nM) CAN01 81.71/84.17 50.04/>100  >100/>100CAN03  5.0/2.90 1.59/1.56 4.12/4.01 Mouse IgG2a, NA/NA NA/NA NA/NAIsotype control Human IgG1, NA/NA NA/NA NA/NA Isotype control

Example N—Analysis of Competitive Binding by ELISA

Protocol

-   -   All samples should be analysed in duplicate.    -   Coat a Nunc-MaxiSorp 96 Micro Well™ Plate with 100 ul/well of        recombinant hIL1RAP 21-367 (1 ug/ml) diluted in 0.01M PBS, pH        7.4.    -   Incubate the plate overnight at 4° C.    -   Wash the plate with ELISA washing buffer        -   (0.01M PBS, 0.05% Tween 20, pH 7.4).    -   Add 150 μl/well of ELISA blocking solution        -   (PBS, 0.5% BSA, 0.05% Tween 20, pH 7.4).    -   Incubate the plate for 1 h at room temperature (RT) under        agitation.    -   Wash the plate with ELISA washing buffer.    -   Add samples of test items (e.g. mAb 1, mAb 2) to wells (100        ul/well, 10 ug/ml)    -   Incubate the plate for 1 h at RT.    -   Wash the plate with ELISA washing solution.    -   Add a solution of reference antibody, such as CAN01 or CAN03        (100 ul/well, 1 ug/ml) to all wells.    -   Incubate the plate for 1 h at RT.    -   Wash the plate with ELISA washing buffer.    -   Add 100 μl/well of a suitable secondary antibody conjugated to        Alkaline rabbit anti-mouse IgG conjugated to Alkaline        Phosphatase (If the test items are human antibodies, a suitable        secondary antibody would be Goat Anti-Mouse IgG (Fc        specific)-Alkaline Phosphatase antibody, SIGMA, A1418)    -   Incubate the plate for 1 h at RT under agitation.    -   Wash the plate with washing buffer.    -   Add 100 μl of pNPP substrate per well.        -   (4-Nitrophenyl phosphatase disodium salt hexahydrate, SIGMA,            1 mg/ml).    -   Incubate the plate at RT under agitation and measure absorbance        at 405 nm consecutively for 30 min. Absorbance at 0 min should        be taken as background signal.

1.-141. (canceled)
 142. An antibody with binding specificity for humaninterleukin-1 receptor accessory protein (IL1RAP) comprising thefollowing complementary determining regions (CDRs): a. Heavy chain CDR1comprising: G F T F S I Y (SEQ ID NO: 21); b. Heavy chain CDR2comprising: S I G G S Y (SEQ ID NO: 22); c. Heavy chain CDR3 comprising:E V D G S Y A M D Y (SEQ ID NO: 23); d. Light chain CDR1 comprising: R AS Q S I G T S I H (SEQ ID NO: 26); e. Light chain CDR2 comprising: S A SE S I S (SEQ ID NO: 27); and f. Light chain CDR3 comprising: Q Q S N S WP T T (SEQ ID NO: 28).
 143. The antibody of claim 142, wherein heavychain CDR1 comprises: G F T F S I Y T M S (SEQ ID NO: 24) and whereinheavy chain CDR2 comprises: T I S I G G S Y I N Y P D S V K G (SEQ IDNO: 25).
 144. An antibody with binding specificity for humaninterleukin-1 receptor accessory protein (IL1RAP) comprising thefollowing complementary determining regions (CDRs): a. Heavy chain CDR1comprising: G F T F S I Y T M S (SEQ ID NO: 24); b. Heavy chain CDR2comprising: T I S I G G S Y I N Y P D S V K G (SEQ ID NO: 25); c. Heavychain CDR3 comprising: E V D G S Y A M D Y (SEQ ID NO: 23); d. Lightchain CDR1 comprising: R A S Q S I G T S I H (SEQ ID NO: 26); e. Lightchain CDR2 comprising: S A S E S I S (SEQ ID NO: 27); and f. Light chainCDR3 comprising: Q Q S N S W P T T (SEQ ID NO: 28).
 145. The antibody ofclaim 142 wherein the antibody exhibits one or more of the followingproperties: a) a binding affinity (K_(D)) for human IL1RAP of 500 pM orgreater; b) cross-reactivity with IL1RAP from Macaca fascicularis; c) aninhibitory action on IL1 signalling; d) capability of inducing ADCC inone or more cancer cell lines; and e) capability of internalisation uponbinding to one or more cancer cell lines.
 146. The antibody of claim142, wherein the antibody is an Fv fragment or an Fab fragment.
 147. Theantibody of claim 142 comprising a heavy chain variable regioncomprising or consisting of the amino acids of SEQ ID NO:
 19. 148. Theantibody of claim 142 comprising a light chain variable regioncomprising or consisting of the amino acids of SEQ ID NO:
 20. 149. Theantibody of claim 142 comprising a heavy chain variable regioncomprising or consisting of amino acids of SEQ ID NO: 19, and a lightchain variable region comprising or consisting of the amino acids of SEQID NO:
 20. 150. The antibody of claim 142 comprising an Fc region. 151.The antibody of claim 142 further comprising a cytotoxic moiety. 152.The antibody of claim 142 further comprising a detectable moiety. 153.An isolated nucleic acid molecule encoding the antibody of claim 142.154. A pharmaceutical composition comprising the antibody of claim 142and a pharmaceutically-acceptable diluent, carrier or excipient.
 155. Amethod for the treatment of a neoplastic disorder in a subject,comprising administering to the subject an effective amount of theantibody of claim 142, wherein the neoplastic disorder is associatedwith cells expressing IL1RAP.
 156. The method of claim 155, wherein theneoplastic disorder is a neoplastic hematologic disorder.
 157. Themethod of claim 156, wherein the neoplastic hematologic disorder isselected from chronic myeloid leukemia (CML), myeloproliferativedisorders (MPD), myelodysplastic syndrome (MDS), acute lymphoblasticleukemia (ALL) and acute myeloid leukemia (AML).
 158. The method ofclaim 155, wherein the neoplastic disorder is associated with theformation of solid tumors within the subject's body.
 159. The method ofclaim 158, wherein the solid tumor is selected from prostate cancer,breast cancer, lung cancer, colorectal cancer, melanomas, bladdercancer, brain/CNS cancer, cervical cancer, oesophageal cancer, gastriccancer, head/neck cancer, kidney cancer, liver cancer, lymphomas,ovarian cancer, pancreatic cancer, and sarcomas.
 160. An in vitro methodfor the detection of cancer cells expressing IL1RAP in a subject, themethod comprising: (a) obtaining cells from a subject to be tested; and(b) detecting whether IL1RAP is present in the cells by contacting thecells with the antibody polypeptide of claim 142, wherein detection ofbinding of the antibody to the cells indicates detection of cancercells.
 161. An in vitro method for identifying a patient with cancer whowould benefit from treatment with the antibody polypeptide of claim 142,comprising: a. obtaining cells from a subject to be tested; b. detectingwhether IL1RAP is present in the cells by contacting the cells with theantibody polypeptide of claim 142, c. determining whether the antibodyor antigen-binding fragment thereof binds to the cells wherein thebinding of the antibody to the cancer cells is indicative of a patientwho would benefit from treatment with the antibody of claim 142.